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OPINION STATEMENT: There are approximately 1.2 million new hematologic malignancy cases resulting in ~ 690,000 deaths each year worldwide, and hematologic malignancies remain the most commonly occurring cancer in children. Even though advances in anticancer treatment regimens in recent decades have considerably improved survival rates, their cytotoxic effects and the resulting long-term complications pose a significant burden on the patients and the health care system. Therefore, non-toxic treatment modalities are needed to decrease side effects. The human body is the host to approximately 40 trillion microbes, known as the human microbiota. The large majority of the microbiota is located in the gastrointestinal tract, and is primarily composed of bacteria. The microbiota plays several important physiological roles, ranging from digestive functions to immunological and neural development. Investigating the microbiota in patients with hematologic malignancies has several important implications. The microbiota affects hematopoiesis, and influences the efficacies of chemotherapy and antimicrobial treatments. Determination of the microbiota composition and diversity could be an important part of risk stratification in the future, and may also take part to personalize antimicrobial treatments. Modulation of the microbiota via probiotics or fecal transplant can potentially be involved in reducing side effects of chemotherapy, and eliminating multiple drug resistant strains in patients with hematologic malignancies.
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Disbiose/etiologia , Neoplasias Hematológicas/complicações , Microbiota , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Bacteriemia/diagnóstico , Bacteriemia/etiologia , Bacteriemia/terapia , Biodiversidade , Terapia Combinada , Gerenciamento Clínico , Disbiose/diagnóstico , Disbiose/terapia , Microbioma Gastrointestinal , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Hospedeiro Imunocomprometido , Metagenoma , Metagenômica/métodosRESUMO
A rapid and accurate detection of pathogens is essential for bedside or on-site diagnosis. Filter paper is an ideal diagnostic tool as it requires no equipment, possesses a high surface-area-to-volume ratio and a high capacity of capillary force. The functionalization of the surface of cellulose filter paper was explored by using glutaric anhydride, N-hydroxysuccinimide, and N,N'-dicyclohexylcarbodiimide. The activated surface systems enable aminated DNA to be immobilized on the surface of filter paper. Both synthetic oligonucleotides and bacterial genomic DNA of Staphylococcus aureus, Escherichia coli, and Campylobacter jejuni were detected successfully. The system produces a clear, consistent and highly visible brown signal within 1-5 min. The digital image can also be analyzed quantitatively due to the brown color resulting from the presence of magnetic beads. Bacterial DNA detection was accomplished by using 16S rDNA probe on the activated paper surface for universal bacterial diagnosis. The method is stable and repeatable. It can detect at least 0.5 pmol of a 120-base synthetic oligonucleotide per assay and 5-10 ng of bacterial DNA per assay. Graphical abstract Schematic representation of the method: a. functionalization of cellulose filter paper, b. printing of aminated probes, c. incubation, d. blocking of unreacted functional groups (as dots shown), e. a visual detection of targets, f. quantitative analysis of image.
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Celulose/química , DNA Bacteriano/análise , Sondas de Oligonucleotídeos/química , Papel , Campylobacter jejuni/isolamento & purificação , Escherichia coli/isolamento & purificação , Tamanho da Partícula , Staphylococcus aureus/isolamento & purificação , Propriedades de SuperfícieRESUMO
Changes in blood glucose concentration alter autonomic function in a manner consistent with altered neural activity in brain regions controlling digestive processes, including neurons in the brain stem nucleus tractus solitarii (NTS), which process viscerosensory information. With whole cell or on-cell patch-clamp recordings, responses to elevating glucose concentration from 2.5 to 15 mM were assessed in identified GABAergic NTS neurons in slices from transgenic mice that express EGFP in a subset of GABA neurons. Single-cell real-time RT-PCR was also performed to detect glutamic acid decarboxylase (GAD67) in recorded neurons. In most identified GABA neurons (73%), elevating glucose concentration from 2.5 to 15 mM resulted in either increased (40%) or decreased (33%) neuronal excitability, reflected by altered membrane potential and/or action potential firing. Effects on membrane potential were maintained when action potentials or fast synaptic inputs were blocked, suggesting direct glucose sensing by GABA neurons. Glucose-inhibited GABA neurons were found predominantly in the lateral NTS, whereas glucose-excited cells were mainly in the medial NTS, suggesting regional segregation of responses. Responses were prevented in the presence of glucosamine, a glucokinase (GCK) inhibitor. Depolarizing responses were prevented when KATP channel activity was blocked with tolbutamide. Whereas effects on synaptic input to identified GABAergic neurons were variable in GABA neurons, elevating glucose increased glutamate release subsequent to stimulation of tractus solitarius in unlabeled, unidentified neurons. These results indicate that GABAergic NTS neurons act as GCK-dependent glucose sensors in the vagal complex, providing a means of modulating central autonomic signals when glucose is elevated.
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Neurônios GABAérgicos/fisiologia , Glucose/metabolismo , Núcleo Solitário/fisiologia , Animais , Fármacos do Sistema Nervoso Central/farmacologia , Feminino , Neurônios GABAérgicos/efeitos dos fármacos , Glucosamina/farmacologia , Glutamato Descarboxilase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipoglicemiantes/farmacologia , Canais KATP/antagonistas & inibidores , Canais KATP/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos Transgênicos , Técnicas de Patch-Clamp , Núcleo Solitário/efeitos dos fármacos , Somatostatina/genética , Somatostatina/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Técnicas de Cultura de Tecidos , Tolbutamida/farmacologiaRESUMO
On-site DNA analysis for diagnostic or forensic purposes is much anticipated in the future of molecular testing. Yet the challenges to achieve this goal remain large with rapid and inexpensive detection and visualization being key factors for any portable analysis system. We have developed a filter paper-based nucleic acid assay, which is able to identify and distinguish dog and human genomic and mitochondrial samples in a forensic setting. The filter paper material allows for transport by capillary force of the sample DNA through the detection surface, allowing the targets to hybridize specifically to their complementary capture sequences. Coupling micrometer-sized beads to DNA allows the results to be visualized by the naked eye, enabling instant, cost-efficient, and on-site detection, while eliminating the need for advanced expensive instrumentation.
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DNA/análise , Filtração/instrumentação , Papel , Animais , DNA/química , DNA/isolamento & purificação , Sondas de DNA/química , Cães , Ciências Forenses , Humanos , Especificidade da Espécie , Propriedades de Superfície , Fatores de TempoRESUMO
Acute lymphocytic leukemia (ALL) is an aggressive hematological malignancy of highly proliferative lymphoblasts. ALL is the most common cancer in children, and is typically treated with combination chemotherapy. The 5-year survival of ALL improved significantly in recent decades with this treatment approach. However, certain age groups (below 2 and over 10 years of age) have much worse prognosis, and over 50% of patients with ALL experience long-term side effects proportional to the dosage of anticancer drugs. Therefore, different treatment strategies are required to improve survival in ALL and to reduce side effects of chemotherapy. Since epigenetic modifications are dominantly reversible, "epidrugs" (drugs targeting epigenetic markers) are considered for feasibility in the treatment of ALL as epigenetic modifications, and acetylation of histones was demonstrated to play a critical role in the pathogenesis of ALL. Histone deacetylases (HDACs) have been shown to be differentially expressed in several hematological malignancies, including ALL. HDAC inhibitors (HDACis) have been shown to express selective toxicity for ALL cells, but they showed limited efficacy and higher than expected toxicity in mouse models or clinical trials in ALL. The aim of this review is to examine the role of the microbiota and microbial metabolites in the mechanisms of HDAC functions, and explore the utilization of the microbiota and microbial metabolites in improving the efficacy of HDACi in ALL. HDAC regulators and natural HDACi are depleted in ALL due to microbiota change leading to a decrease in butyrate and propionate, and HDACi treatment is not effective in ALL due to their short half-life. We propose that HDACi released by the microbiota may be necessary in HDAC regulation and this process is impaired in ALL. Furthermore, the review will also consider the role of restoration of the microbiota or supplementation of natural HDACi in potentially restoring HDAC and HDACi functions.
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Background: Social isolation (SI) and loneliness are major adult and adolescent health concerns, particularly in the coronavirus disease 2019 (COVID-19) era. Recent prospective cohort studies indicate that older women who experienced both SI and loneliness had a significantly higher risk of cardiovascular disease (CVD). Hypertension, a well-established risk factor for CVD, is more prevalent in elderly women than men. Furthermore, a lack of social relationships is strongly associated with an increased risk of hypertension in middle-aged and elderly women compared to men. Although this has not been extensively studied, adolescents and young adults who experience loneliness or SI may also be at risk for CVD and depression. The purpose of this study was to examine the effect of SI on blood pressure and depression-like behavior in young male and female mice. Methods: Weaned C57BL/6 mice were randomly assigned (n = 6/group/sex) to either group housing (GH) or SI. Animals in the SI group were housed in individual cages for 8 weeks with no view of other animals. The cages were kept in ventilated racks to prevent pheromone exposure and socially isolated animals had no cage enrichment. Results: SI increased systolic, diastolic, and mean arterial blood pressure in females and elevated heart rate in both sexes. Body weight gain was dramatically increased in socially isolated females but tended to decrease in socially isolated males. In the forced swim test, which detects depression-like behavior, there was no difference between groups in total immobility time. The latency to immobility, however, was significantly decreased in socially isolated females. Serum concentrations of corticosterone and metanephrine did not differ between socially isolated and group-housed females, but corticosterone levels were significantly reduced in socially isolated males. Conclusions: Our results indicate that 8 weeks of SI leads to significant changes in blood pressure and heart rate and mild changes in depression-like behavior in young mice, with females affected more than males.
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Bloodstream infection (BSI) is a major complication in patients with cancers due to therapy-induced neutropenia and underlying conditions, which increases hospitalization time and mortality rate. Targeted and timely antimicrobial management is crucial to save the patients' lives and reduce the social and economic burdens. Blood culture is a routine clinical diagnostic method of BSI with a long turnaround time, and generally identifies monomicrobial BSI. Thus, polymicrobial BSI often goes undetected although it occurs more frequently in these patients and results in more severe outcomes compared to monomicrobial BSI. In this work, we apply glutaric anhydride, N-hydroxysuccinimide and N,N'-dicyclohexylcarbodiimide to fabricate a functional surface on cellulose filter paper. Targeting three pathogens (Escherichia coli, Saccharomyces cerevisiae, and human cytomegalovirus) commonly occurring in BSI in neutropenic patients, we demonstrate rapid and accurate triplex pathogen DNA detection using the functionalized paper. All three pathogen DNA was identified in 1-5 min with a detection limit of 0.1-0.5 ng/µL. The developed test tool has the potential to provide rapid polymicrobial BSI diagnosis in support of timely, accurate antimicrobial treatment, and could be integrated into an automatic sample-to-result portable equipment.
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Bacteriemia , Coinfecção , Sepse , Antibacterianos/farmacologia , Bacteriemia/diagnóstico , Hemocultura , DNA , Escherichia coli/genética , HumanosRESUMO
Background: Hematological malignancies are the most common cancers in the pediatric population, and T-cell acute lymphocytic leukemia (T-ALL) is the most common hematological malignancy in children. Bloodstream infection (BSI) is a commonly occurring complication in leukemia due to underlying conditions and therapy-induced neutropenia. Several studies identified the gut microbiome as a major source of BSI due to bacterial translocation. This study aimed to investigate changes in the intestinal and fecal microbiome, and their roles in the pathophysiology of BSI in a pediatric T-ALL mouse model using high-throughput shotgun metagenomics sequencing, and metabolomics. Results: Our results show that BSI in ALL is characterized by an increase of a mucin degrading bacterium (Akkermansia muciniphila) and a decrease of butyrate producer Clostridia spp., along with a decrease in short-chain fatty acid (SCFA) concentrations and differential expression of tight junction proteins in the small intestine. Functional analysis of the small intestinal microbiome indicated a reduced capability of SCFA synthesis, while SCFA supplementation ameliorated the development of BSI in ALL. Conclusions: Our data indicates that changes in the microbiome, and the resulting changes in levels of SCFAs contribute significantly to the pathogenesis of bloodstream infection in ALL. Our study provides tailored preventive or therapeutic approaches to reduce BSI-associated mortality in ALL.
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BACKGROUND: Limited studies associate changes in microbiota composition and metabolites among children and adolescents with obesity. Decreases in compositional diversity, increases in the proportion of Firmicutes and Bacteroidetes (F/B ratio) and increases in short-chain fatty acids (SCFAs) have been proposed as contributing factors in the pathophysiology of obesity. OBJECTIVES: The aim of the current study was to characterize the faecal microbiota composition, diversity, F/B ratio and SCFA levels in different weight categories (lean, overweight, obesity classes 1-3) of children ages 5 to 12 years. METHODS: We collected and processed 83 samples from different weight categories (27.7% lean, 11% overweight, 15%, 17% and 17% of obesity classes 1, 2, and 3, respectively). Microbiota content was determined by sequencing the V4 region of the 16S rRNA gene, and SCFA content was analyzed. RESULTS: Microbiota compositions showed no significant differences in diversity or F/B ratios between weight categories. However, a relative abundance of Proteobacteria and lack of Verrucomicrobia were demonstrated when comparing severe obesity to the leaner groups. Faecal butyrate, propionate and isopentanoate concentrations increased progressively with weight category demonstrating significance in the class 3 obesity group. CONCLUSIONS: Our results show that severe childhood obesity in our study population was associated with changes in gut microbiome composition correlated to previously reported cardiometabolic disease states in obesity. Increased SCFA levels correlate with obesity-related microbiome metabolic function without a reduction in diversity characterized at a phyla level. Further characterization of these specimens at a species level and longitudinal studies are needed to elucidate these relationships.
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Microbioma Gastrointestinal , Obesidade Infantil , Adolescente , Criança , Pré-Escolar , Estudos Transversais , Ácidos Graxos Voláteis , Fezes , Humanos , RNA Ribossômico 16S/genéticaRESUMO
Hematological malignancies are the most common type of pediatric cancers, and acute lymphocytic leukemia (ALL) is the most frequently occurring hematological malignancy during childhood. A major cause of mortality in leukemia is bloodstream infection (BSI). The aim of the current study was to explore the gut microbiota in ALL and its potential functional alterations. High-throughput sequencing was used to characterize the bacterial and fungal microbiota in feces and their predicted functional characteristics in a xenotransplant pediatric ALL mouse model. Our work shows that gut microbiota significantly changes in leukemia, which may result in functional alterations. This study may provide potential therapeutic or preventive strategies of BSI in ALL.
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Microbiota , Leucemia-Linfoma Linfoblástico de Células Precursoras/microbiologia , Animais , Biodiversidade , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal , Sequenciamento de Nucleotídeos em Larga Escala , Metagenômica/métodos , Camundongos , RNA Ribossômico 16SRESUMO
Point-of-care (POC) detection is crucial in clinical diagnosis in order to provide timely and specific treatment. Combining polyamidoamine (PAMAM) dendrimer, p-phenylene diisothiocyanate (PDITC) and superparamagnetic beads, a novel method to activate the surface of filter paper to bind DNA molecules has been developed. The method is based on the primary amination of the filter paper surface with PAMAM dendrimer, followed by generation of isothiocyanate groups via PDITC, and subsequent repetition of these two steps. Different parameters of the process have been optimized, including probe printing, preparation of target DNAs and detection. The result shows that, due to the highly porous structure of filter paper, high amounts of printed probes, target DNAs and magnetic beads can provide high signal intensities in the detection area via probe/target duplex formation. This method is suitable for rapid, specific and cost-efficient DNA detection on cellulose filter paper. It can be used as a POC device, in particular for diagnosis and treatment management of infectious diseases and identification of antimicrobial drug resistance genes.
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DNA/análise , Filtração , Papel , Celulose/química , Dendrímeros/química , Limite de Detecção , Magnetismo , Metanol/química , Microesferas , Poliaminas/química , Propriedades de Superfície , Tiocianatos/químicaRESUMO
In order to provide additional information to the epidemiological situation in Middle Europe and open further possibilities to investigate the transmission of influenza viruses between species, the viral genomes of three influenza A virus isolates (one human and two swine) collected from North-East Hungary in 20062007 have been fully sequenced and characterized. The sequence analysis reveals strong geographical relationships between the internal genes of the two swine viruses; the human isolate shows strict conservation to recent H1N1 strains, while the swine strains demonstrate and reflect a mixed avianhuman origin, a characteristic of European swine influenza viruses. No evidence of interspecies interaction has been found among the studied isolates.
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Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Influenza Humana/virologia , Infecções por Orthomyxoviridae/veterinária , Doenças dos Suínos/virologia , Animais , Análise por Conglomerados , Genoma Viral , Humanos , Hungria , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H3N2/classificação , Dados de Sequência Molecular , Infecções por Orthomyxoviridae/virologia , Filogeografia , RNA Viral/genética , Análise de Sequência de DNA , Homologia de Sequência , SuínosRESUMO
Bloodstream infection (BSI) is a life-threatening condition characterized by the presence of pathogens in the blood. It is associated with increased morbidity and mortality, and has to be treated promptly as mortality increases with every hour of delayed treatment. Therefore, rapid and sensitive diagnosis of BSI is essential. The routine diagnostic method for BSI is blood culture, which can only detect culturable pathogens and takes several days to obtain results. The 16S rRNA gene is present in all bacteria and is commonly used as a target for universal bacterial detection in rapid molecular assays such as PCR. However, molecular detection of the 16S gene is hampered by the large amount of human DNA found in blood samples, making diagnostic results aspecific and less sensitive. We have optimized the selection of PCR primers targeting the 16S rRNA gene to avoid cross-reaction with human DNA background. The developed method increases specificity and sensitivity for pathogen diagnosis, and provides rapid and accurate pathogen detection for rare bacterial DNA in the presence of abundant host DNA.
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Bacteriemia/diagnóstico , Bactérias/isolamento & purificação , Primers do DNA/genética , RNA Ribossômico 16S/genética , Bactérias/classificação , Bactérias/genética , DNA Bacteriano/genética , DNA Ribossômico/genética , Diagnóstico Precoce , Humanos , Sensibilidade e Especificidade , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
Bloodstream infection (BSI) is the major cause of mortality in acute lymphocytic leukemia (ALL). Causative pathogens in BSI originate from the gut microbiota due to an increase in intestinal permeability, a process known as bacterial translocation (BT). The gut microbiota in physiological conditions is controlled by a large number of immune cells as part of the gut-associated lymphoid tissue (GALT).The aim of the current study was to investigate the mechanism of bacterial translocation in leukemia by identifying and characterizing alterations in the GALT in leukemic mouse model. Our studies revealed a severe impairment of the GALT characterized by a loss of lymphatic cells in ALL, which eventually led to BSI. We identified differentially expressed genes in the intraepithelium and the lamina propria, which may contribute to BT and to the impairment of lymphocyte migration.
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Translocação Bacteriana , Microbioma Gastrointestinal , Mucosa Intestinal/microbiologia , Tecido Linfoide/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/microbiologia , Animais , Linhagem Celular Tumoral , Movimento Celular , Feminino , Perfilação da Expressão Gênica , Histonas/química , Humanos , Imunidade nas Mucosas , Linfócitos/citologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Domínios Proteicos , RNA Ribossômico 16S/genética , Análise de Sequência de RNARESUMO
A subtyping assay for both the hemagglutinin (HA) and neuraminidase (NA) surface antigens of the avian influenza virus (AIV) has been developed. The method uses padlock probe chemistry combined with a microarray output for detection. The outstanding feature of this assay is its capability to designate both the HA and the NA of an AIV sample from a single reaction mixture. A panel of 77 influenza virus strains was tested representing the entire assortment of the two antigens. One hundred percent (77/77) of the samples tested were identified as AIV, and 97% (75/77) were subtyped correctly in accordance with previous examinations performed by classical diagnostic methods. Testing of heterologous pathogens verified the specificity of the assay. This assay is a convenient and practical tool for the study of AIVs, providing important HA and NA data more rapidly than conventional methods.
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Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Influenza Aviária/virologia , Neuraminidase/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Orthomyxoviridae/classificação , Orthomyxoviridae/genética , Animais , Primers do DNA/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Aves Domésticas , Sensibilidade e Especificidade , Proteínas Virais/genéticaRESUMO
BACKGROUND: Although the important role of the non-structural 1 (NS) gene of influenza A in virulence of the virus is well established, our knowledge about the extent of variation in the NS gene pool of influenza A viruses in their natural reservoirs in Europe is incomplete. In this study we determined the subtypes and prevalence of influenza A viruses present in mallards in Northern Europe and further analysed the NS gene of these isolates in order to obtain a more detailed knowledge about the genetic variation of NS gene of influenza A virus in their natural hosts. RESULTS: A total number of 45 influenza A viruses of different subtypes were studied. Eleven haemagglutinin- and nine neuraminidase subtypes in twelve combinations were found among the isolated viruses. Each NS gene reported here consisted of 890 nucleotides; there were no deletions or insertions. Phylogenetic analysis clearly shows that two distinct gene pools, corresponding to both NS allele A and B, were present at the same time in the same geographic location in the mallard populations in Northern Europe. A comparison of nucleotide sequences of isolated viruses revealed a substantial number of silent mutations, which results in high degree of homology in amino acid sequences. The degree of variation within the alleles is very low. In our study allele A viruses displays a maximum of 5% amino acid divergence while allele B viruses display only 2% amino acid divergence. All the viruses isolated from mallards in Northern Europe possessed the typical avian ESEV amino acid sequence at the C-terminal end of the NS1 protein. CONCLUSION: Our finding indicates the existence of a large reservoir of different influenza A viruses in mallards population in Northern Europe. Although our phylogenetic analysis clearly shows that two distinct gene pools, corresponding to both NS allele A and B, were present in the mallards populations in Northern Europe, allele B viruses appear to be less common in natural host species than allele A, comprising only about 13% of the isolates sequenced in this study.
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Anseriformes/virologia , Vírus da Influenza A/classificação , Vírus da Influenza A/isolamento & purificação , Filogenia , Proteínas não Estruturais Virais/genética , Animais , Europa (Continente) , Vírus da Influenza A/genética , Dados de Sequência Molecular , Mutação , Homologia de SequênciaRESUMO
BACKGROUND: The analysis of the nonstructural (NS) gene of the highly pathogenic (HP) H5N1 avian influenza viruses (AIV) isolated in Sweden early 2006 indicated the co-circulation of two sub-lineages of these viruses at that time. In order to complete the information on their genetic features and relation to other HP H5N1 AIVs the seven additional genes of twelve Swedish isolates were amplified in full length, sequenced, and characterized. RESULTS: The presence of two sub-lineages of HP H5N1 AIVs in Sweden in 2006 was further confirmed by the phylogenetic analysis of approximately the 95% of the genome of twelve isolates that were selected on the base of differences in geographic location, timing and animal species of origin. Ten of the analyzed viruses belonged to sub-clade 2.2.2. and grouped together with German and Danish isolates, while two 2.2.1. sub-clade viruses formed a cluster with isolates of Egyptian, Italian, Slovenian, and Nigerian origin. The revealed amino acid differences between the two sub-groups of Swedish viruses affected the predicted antigenicity of the surface glycoproteins, haemagglutinin and neuraminidase, rather than the nucleoprotein, polymerase basic protein 2, and polymerase acidic protein, the main targets of the cellular immune responses. The distinctive characteristics between members of the two subgroups were identified and described. CONCLUSION: The comprehensive genetic characterization of HP H5N1 AIVs isolated in Sweden during the spring of 2006 is reported. Our data support previous findings on the coincidental spread of multiple sub-lineage H5N1 HPAIVs via migrating aquatic birds to large distance from their origin. The detection of 2.2.1. sub-clade viruses in Sweden adds further data regarding their spread in the North of Europe in 2006. The close genetic relationship of Swedish isolates sub-clade 2.2.2. to the contemporary German and Danish isolates supports the proposition of the introduction and spread of a single variant of 2.2.2. sub-clade H5N1 avian influenza viruses in the Baltic region. The presented findings underline the importance of whole genome analysis.
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Virus da Influenza A Subtipo H5N1/classificação , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/virologia , Animais , Aves , Análise por Conglomerados , Genoma Viral , Virus da Influenza A Subtipo H5N1/genética , Influenza Aviária/epidemiologia , Epidemiologia Molecular , Dados de Sequência Molecular , Filogenia , RNA Viral/genética , Análise de Sequência de DNA , Homologia de Sequência , Suécia/epidemiologia , Proteínas não Estruturais Virais/genética , Proteínas Estruturais Virais/genéticaRESUMO
Hepatitis E virus (HEV) is a major cause of food- and waterborne diseases in countries with poor sanitation. Furthermore, travellers to such countries are also at risk of contracting the virus. Noteworthily, during the last decade an increasing number of non-travel-related cases were recorded even in countries with high sanitary standards. An alternative, direct route of infection, from animals to humans (zoonotic transmission) is suspected to be the cause of recent cases of hepatitis E. In order to provide rapid and sensitive methods for detecting the virus in various hosts, two real-time PCR methods were developed and compared: a TaqMan and Primer-Probe Energy Transfer (PriProET) assay. These highly sensitive novel methods provide valuable diagnostic tools to investigate zoonotic transmission, to detect the virus in the food chain and in research related to the potential of hepatitis E virus to cross the species barrier. The results show that the two novel PCR assays are robust, highly sensitive and specific for broad range detection of the four genotypes of HEV. Compared to PriProET, the TaqMan assay appears to perform slightly better, with higher fluorescence values for positive samples. However, the PriProET has the benefit of better tolerating the point mutations in the target nucleic acids. Thus, it provides a more powerful tool to detect new virus variants. These new molecular diagnostic assays are practical tools that can be employed in the area of public health, for disease diagnosis and for tracking outbreaks. In basic research the methods provide new tools to study HEV biology, including virus-host interactions and transmission between various host species.
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Vírus da Hepatite E/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Animais , Sequência de Bases , Hepatite E/diagnóstico , Hepatite E/virologia , Humanos , Macaca mulatta/virologia , Dados de Sequência Molecular , Sensibilidade e Especificidade , Suínos/virologiaRESUMO
The World Organization for Animal Health (Office International des Epizooties, OIE) includes the diseases caused by foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), and vesicular stomatitis virus (VSV), as "Diseases Notifiable to the OIE". Foot-and-mouth disease (FMD) outbreaks have severe economical as well as social effects and cannot be differentiated from the diseases caused by the other two viruses on the basis of clinical symptoms. Efficient laboratory techniques are therefore required for detection and identification of the viruses causing similar vesicular symptoms in swine. A rapid method is described using padlock probes and microarrays to detect simultaneously and differentiate the three viruses in a single reaction, as well as providing serotype information in cases of VSV infection. The padlock probe/microarray assay detected successfully and identified 39 cDNA samples of different origin representing the three viruses. The results were in complete agreement with identities and serotypes determined previously. This novel virus detection method is discussed in terms of usefulness and further development.