Péter Gyarmati

Genetic characterization of the NS gene indicates co-circulation of two sub-lineages of highly pathogenic avian influenza virus of H5N1 subtype in Northern Europe in 2006

The non-structural (NS) gene of highly pathogenic avian influenza viruses of the H5N1 subtype (HPAI-H5N1) isolated in Baltic Sea area of Sweden in 2006 was studied. The phylogenetic analysis data demonstrated that two distinct sub-lineages of HPAI-H5N1 were circulating during the outbreak in Northern Europe in Spring 2006. Sub-lineage I viruses fell into the same clade as viruses found in Denmark and Germany and formed a sub-clade which also included viruses isolated in the Russian Federation in late 2005. Sub-lineage II viruses formed a sub-clade closely related to European, Middle Eastern and African isolates reported in 2006. Analysis of the inferred amino acid sequences of the NS1 protein showed a deletion of five amino acids at positions 80–84. No viruses represented in this study contained Glu92 in the NS1 and all isolates contained the avian-like ESKV amino acid sequences at the NS1 C-terminal end. Sub-lineage I isolates contained unique substitutions V194I in NS1 and G63E in Nuclear export protein (NEP).

Molecular characterization of highly pathogenic H5N1 avian influenza viruses isolated in Sweden in 2006

BackgroundThe 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.ResultsThe 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.ConclusionThe 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.

Visual detection of DNA on paper chips

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.

Simultaneous Genotyping of All Hemagglutinin and Neuraminidase Subtypes of Avian Influenza Viruses by Use of Padlock Probes

ABSTRACT 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.

Phylogenetic analysis of the non-structural (NS) gene of influenza A viruses isolated from mallards in Northern Europe in 2005

BackgroundAlthough 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.ResultsA 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.ConclusionOur 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.

The rapid molecular subtyping and pathotyping of avian influenza viruses.

Highly conserved nucleotide stretches flanking the cleavage site of the haemagglutinin (HA) gene of influenza type A viruses were utilised for generating PCR amplicons from a broad range of avian influenza viruses (AIV) in a one-step real-time SYBR Green RT-PCR assay. The nucleotide sequencing of the amplified PCR products simultaneously reveals both the HA subtype and the pathotype of the AIV isolates, as we demonstrated in case of H5 subtype viruses. The specificity of the assay was confirmed by investigating 66 strains of AIV and nine heterologous pathogens, including influenza B, C and various avian pathogenic viruses. This assay enables a general HA subtype identification and pathotype determination of AIV isolates providing a useful alternative tool for avian influenza diagnosis.

Metagenomic analysis of bloodstream infections in patients with acute leukemia and therapy-induced neutropenia

Leukemic patients are often immunocompromised due to underlying conditions, comorbidities and the effects of chemotherapy, and thus at risk for developing systemic infections. Bloodstream infection (BSI) is a severe complication in neutropenic patients, and is associated with increased mortality. BSI is routinely diagnosed with blood culture, which only detects culturable pathogens. We analyzed 27 blood samples from 9 patients with acute leukemia and suspected BSI at different time points of their antimicrobial treatment using shotgun metagenomics sequencing in order to detect unculturable and non-bacterial pathogens. Our findings confirm the presence of bacterial, fungal and viral pathogens alongside antimicrobial resistance genes. Decreased white blood cell (WBC) counts were associated with the presence of microbial DNA, and was inversely proportional to the number of sequencing reads. This study could indicate the use of high-throughput sequencing for personalized antimicrobial treatments in BSIs.

Bacterial Landscape of Bloodstream Infections in Neutropenic Patients via High Throughput Sequencing

Background Bloodstream infection (BSI) is a common and potentially life-threatening complication in patients with hematological malignancies and therapy-induced neutropenia. Administration of broad spectrum antibiotics has substantially decreased the mortality rate in febrile neutropenia, but bacterial infection is documented in only one-third or fewer of the cases. BSI is typically diagnosed by blood culture; however, this method can detect only culturable pathogens. Methods In the present study, a total of 130 blood samples from hematological patients receiving dose-intensive antitumoural treatment were subjected to 16S rRNA PCR and 62 of them were cultured. PCR positive samples were processed to high throughput sequencing by amplifying the V1-V3 regions of the 16S rRNA gene to obtain a full spectrum of bacteria present in BSI. Results Five phyla and 30 genera were identified with sequencing compared to 2 phyla and 4 genera with culture. The largest proportion of bacteria detected by sequencing belonged to Proteobacteria (55.2%), Firmicutes (33.4%) and Actinobacteria (8.6%), while Fusobacteria (0.4%) and Bacteroidetes (0.1%) were also detected. Ninety-eight percent of the bacteria identified by sequencing were opportunistic human pathogens and 65% belonged to the normal human microbiota. Conclusions The present study indicates that BSIs in neutropenic hosts contain a much broader diversity of bacteria, likely with host origin, than previously realized. The elevated ratio of Proteobacteria in BSI corroborates the results found in other systemic inflammatory diseases, such as inflammatory bowel disease or mucosal infections. This knowledge may become of value for tailoring antimicrobial drug administration.

Glucose sensing by GABAergic neurons in the mouse nucleus tractus solitarii.

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.

Nuclease-Assisted Suppression of Human DNA Background in Sepsis

Sepsis is a severe medical condition characterized by a systemic inflammatory response of the body caused by pathogenic microorganisms in the bloodstream. Blood or plasma is typically used for diagnosis, both containing large amount of human DNA, greatly exceeding the DNA of microbial origin. In order to enrich bacterial DNA, we applied the C0t effect to reduce human DNA background: a model system was set up with human and Escherichia coli (E. coli) DNA to mimic the conditions of bloodstream infections; and this system was adapted to plasma and blood samples from septic patients. As a consequence of the C0t effect, abundant DNA hybridizes faster than rare DNA. Following denaturation and re-hybridization, the amount of abundant DNA can be decreased with the application of double strand specific nucleases, leaving the non-hybridized rare DNA intact. Our experiments show that human DNA concentration can be reduced approximately 100,000-fold without affecting the E. coli DNA concentration in a model system with similarly sized amplicons. With clinical samples, the human DNA background was decreased 100-fold, as bacterial genomes are approximately 1,000-fold smaller compared to the human genome. According to our results, background suppression can be a valuable tool to enrich rare DNA in clinical samples where a high amount of background DNA can be found.