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Find video protocols related to scientific articles indexed in Pubmed.
Detection of rotavirus using padlock probes and rolling circle amplification.
PLoS ONE
PUBLISHED: 01-01-2014
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Rotavirus infections are one of the most common reasons for hospitalizations due to gastrointestinal diseases. Rotavirus is often diagnosed by latex agglutination assay, chromatography immunoassay, or by electron microscopy, which are all quite insensitive. Reverse transcription polymerase chain reaction, on the other hand, is very sensitive to variations at the genomic level. We developed a novel assay based on a set of 58 different padlock probes with a detection limit of 1,000 copies. Twenty-two patient samples were analyzed and the assay showed high concordance with a PCR-based assay. In summary, we present a new assay for sensitive and variation tolerant detection of rotavirus.
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Transmission of hepatitis C virus among intravenous drug users in the Uppsala region of Sweden.
Infect Ecol Epidemiol
PUBLISHED: 01-01-2014
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Epidemiology and transmission patterns of hepatitis C virus (HCV) are important subjects as we enter a new era of treatment with directly acting antivirals (DAAs). The highest prevalence of HCV in developed countries is found among intravenous drug users (IDUs), where unsafe needle sharing practices provide the main route of infection. Efforts to prohibit the continuous spread of HCV among these groups have been initiated by the community services and health care providers. Our goal was to understand how HCV was transmitted among IDUs within a limited population group. We provide a retrospective study (2005-2007) of the HCV transmission patterns in a population of IDUs in the Uppsala region of Sweden.
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Quantitative fucK gene polymerase chain reaction on sputum and nasopharyngeal secretions to detect Haemophilus influenzae pneumonia.
Diagn. Microbiol. Infect. Dis.
PUBLISHED: 02-08-2013
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A quantitative polymerase chain reaction (PCR) for the fucK gene was developed for specific detection of Haemophilus influenzae. The method was tested on sputum and nasopharyngeal aspirate (NPA) from 78 patients with community-acquired pneumonia (CAP). With a reference standard of sputum culture and/or serology against the patients own nasopharyngeal isolate, H. influenzae etiology was detected in 20 patients. Compared with the reference standard, fucK PCR (using the detection limit 10(5) DNA copies/mL) on sputum and NPA showed a sensitivity of 95.0% (19/20) in both cases, and specificities of 87.9% (51/58) and 89.5% (52/58), respectively. In a receiver operating characteristic curve analysis, sputum fucK PCR was found to be significantly superior to sputum P6 PCR for detection of H. influenzae CAP. NPA fucK PCR was positive in 3 of 54 adult controls without respiratory symptoms. In conclusion, quantitative fucK real-time PCR provides a sensitive and specific identification of H. influenzae in respiratory secretions.
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Conserved structure and inferred evolutionary history of long terminal repeats (LTRs).
Mob DNA
PUBLISHED: 02-01-2013
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Long terminal repeats (LTRs, consisting of U3-R-U5 portions) are important elements of retroviruses and related retrotransposons. They are difficult to analyse due to their variability.The aim was to obtain a more comprehensive view of structure, diversity and phylogeny of LTRs than hitherto possible.
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Quantitative and multiplex detection of pathogenic fungi using padlock probes, generic qPCR, and suspension array readout.
Methods Mol. Biol.
PUBLISHED: 01-09-2013
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The multiplexing qualities of padlock probes and Luminex™ technology combined with the well-established quantitative feature of qPCR were the base for a ten-plex fungal detection protocol that quantitatively reveals ten different fungal species in a single experiment. Padlock probes are oligonucleotides designed to form circular DNA when hybridizing to specific target DNA. The 5 and 3 regions of the probes meet and ligate only when a specific target sequence is present in the examined sample. The region of the padlock probes that separates the target-specific 5 and 3 ends contains general primer sequences for amplification of circularized probes by means of rolling circle amplification (RCA) and qPCR. The interspersed region also contains specific tag sequences for subsequent Luminex™ recognition.
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Epitopes of microbial and human heat shock protein 60 and their recognition in myalgic encephalomyelitis.
PLoS ONE
PUBLISHED: 01-01-2013
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Myalgic encephalomyelitis (ME, also called Chronic Fatigue Syndrome), a common disease with chronic fatigability, cognitive dysfunction and myalgia of unknown etiology, often starts with an infection. The chaperonin human heat shock protein 60 (HSP60) occurs in mitochondria and in bacteria, is highly conserved, antigenic and a major autoantigen. The anti-HSP60 humoral (IgG and IgM) immune response was studied in 69 ME patients and 76 blood donors (BD) (the Training set) with recombinant human and E coli HSP60, and 136 30-mer overlapping and targeted peptides from HSP60 of humans, Chlamydia, Mycoplasma and 26 other species in a multiplex suspension array. Peptides from HSP60 helix I had a chaperonin-like activity, but these and other HSP60 peptides also bound IgG and IgM with an ME preference, theoretically indicating a competition between HSP60 function and antibody binding. A HSP60-based panel of 25 antigens was selected. When evaluated with 61 other ME and 399 non-ME samples (331 BD, 20 Multiple Sclerosis and 48 Systemic Lupus Erythematosus patients), a peptide from Chlamydia pneumoniae HSP60 detected IgM in 15 of 61 (24%) of ME, and in 1 of 399 non-ME at a high cutoff (p<0.0001). IgM to specific cross-reactive epitopes of human and microbial HSP60 occurs in a subset of ME, compatible with infection-induced autoimmunity.
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The evolution of infectious agents in relation to sex in animals and humans: brief discussions of some individual organisms.
Ann. N. Y. Acad. Sci.
PUBLISHED: 08-10-2011
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The following series of concise summaries addresses the evolution of infectious agents in relation to sex in animals and humans from the perspective of three specific questions: (1) what have we learned about the likely origin and phylogeny, up to the establishment of the infectious agent in the genital econiche, including the relative frequency of its sexual transmission; (2) what further research is needed to provide additional knowledge on some of these evolutionary aspects; and (3) what evolutionary considerations might aid in providing novel approaches to the more practical clinical and public health issues facing us currently and in the future?
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Rational recombinant XMRV antigen preparation and bead coupling for multiplex serology in a suspension array.
Protein Expr. Purif.
PUBLISHED: 06-18-2011
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Diagnosis of infectious diseases often requires demonstration of antibodies to the microbe (serology). A large set of antigens, covering viruses, bacteria, fungi and parasites may be needed. Recombinant proteins have a prime role in serological tests. Suspension arrays offer high throughput for simultaneous measurement of many different antibodies. We here describe a rational process for preparation, purification and coupling to beads of recombinant proteins prepared in Escherichia coli derivate Origami B, to be used in a serological Luminex suspension array. All six Gag and Env proteins (p10, p12, p15, p30, gp70 and p15E), from the xenotropic murine leukemia virus-related virus (XMRV), were prepared, allowing the creation of a multiepitope XMRV antibody assay. The procedure is generic and allows production of protein antigens ready for serological testing in a few working days. Instability and aggregation problems were circumvented by expression of viral proteins fused to a carrier protein (thioredoxin A; TrxA), purification via inclusion body formation, urea solubilization, His tag affinity chromatography and direct covalent coupling to microspheres without removal of the elution buffer. The yield of one preparation (2-10mg fusion protein per 100ml culture) was enough for 20-100 coupling reactions, sufficing for tests of many tens of thousands of sera. False serological positivity due to antibodies binding to TrxA and to traces of E. coli proteins remaining in the preparation could be reduced by preabsorption of sera with free TrxA and E. coli extract. The recombinant antigens were evaluated using anti-XMRV antibodies. Although hybrid proteins expressed in E. coli in this way will not have the entire tertiary structure and posttranslational modifications of the native proteins, they contain a large subset of the epitopes associated with them. The described strategy is simple, quick, efficient and cheap. It should be applicable for suspension array serology in general.
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Murine gammaretrovirus group G3 was not found in Swedish patients with myalgic encephalomyelitis/chronic fatigue syndrome and fibromyalgia.
PLoS ONE
PUBLISHED: 05-11-2011
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The recent report of gammaretroviruses of probable murine origin in humans, called xenotropic murine retrovirus related virus (XMRV) and human murine leukemia virus related virus (HMRV), necessitated a bioinformatic search for this virus in genomes of the mouse and other vertebrates, and by PCR in humans.
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The first sequenced carnivore genome shows complex host-endogenous retrovirus relationships.
PLoS ONE
PUBLISHED: 04-18-2011
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Host-retrovirus interactions influence the genomic landscape and have contributed substantially to mammalian genome evolution. To gain further insights, we analyzed a female boxer (Canis familiaris) genome for complexity and integration pattern of canine endogenous retroviruses (CfERV). Intriguingly, the first such in-depth analysis of a carnivore species identified 407 CfERV proviruses that represent only 0.15% of the dog genome. In comparison, the same detection criteria identified about six times more HERV proviruses in the human genome that has been estimated to contain a total of 8% retroviral DNA including solitary LTRs. These observed differences in man and dog are likely due to different mechanisms to purge, restrict and protect their genomes against retroviruses. A novel group of gammaretrovirus-like CfERV with high similarity to HERV-Fc1 was found to have potential for active retrotransposition and possibly lateral transmissions between dog and human as a result of close interactions during at least 10.000 years. The CfERV integration landscape showed a non-uniform intra- and inter-chromosomal distribution. Like in other species, different densities of ERVs were observed. Some chromosomal regions were essentially devoid of CfERVs whereas other regions had large numbers of integrations in agreement with distinct selective pressures at different loci. Most CfERVs were integrated in antisense orientation within 100 kb from annotated protein-coding genes. This integration pattern provides evidence for selection against CfERVs in sense orientation relative to chromosomal genes. In conclusion, this ERV analysis of the first carnivorous species supports the notion that different mammals interact distinctively with endogenous retroviruses and suggests that retroviral lateral transmissions between dog and human may have occurred.
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Phylogeny-directed search for murine leukemia virus-like retroviruses in vertebrate genomes and in patients suffering from myalgic encephalomyelitis/chronic fatigue syndrome and prostate cancer.
Adv Virol
PUBLISHED: 04-17-2011
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Gammaretrovirus-like sequences occur in most vertebrate genomes. Murine Leukemia Virus (MLV) like retroviruses (MLLVs) are a subset, which may be pathogenic and spread cross-species. Retroviruses highly similar to MLLVs (xenotropic murine retrovirus related virus (XMRV) and Human Mouse retrovirus-like RetroViruses (HMRVs)) reported from patients suffering from prostate cancer (PC) and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) raise the possibility that also humans have been infected. Structurally intact, potentially infectious MLLVs occur in the genomes of some mammals, especially mouse. Mouse MLLVs contain three major groups. One, MERV G3, contained MLVs and XMRV/HMRV. Its presence in mouse DNA, and the abundance of xenotropic MLVs in biologicals, is a source of false positivity. Theoretically, XMRV/HMRV could be one of several MLLV transspecies infections. MLLV pathobiology and diversity indicate optimal strategies for investigating XMRV/HMRV in humans and raise ethical concerns. The alternatives that XMRV/HMRV may give a hard-to-detect "stealth" infection, or that XMRV/HMRV never reached humans, have to be considered.
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A revised nomenclature for transcribed human endogenous retroviral loci.
Mob DNA
PUBLISHED: 02-11-2011
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Endogenous retroviruses (ERVs) and ERV-like sequences comprise 8% of the human genome. A hitherto unknown proportion of ERV loci are transcribed and thus contribute to the human transcriptome. A small proportion of these loci encode functional proteins. As the role of ERVs in normal and diseased biological processes is not yet established, transcribed ERV loci are of particular interest. As more transcribed ERV loci are likely to be identified in the near future, the development of a systematic nomenclature is important to ensure that all information on each locus can be easily retrieved.
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Hybridization properties of long nucleic acid probes for detection of variable target sequences, and development of a hybridization prediction algorithm.
Nucleic Acids Res.
PUBLISHED: 09-22-2010
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One of the main problems in nucleic acid-based techniques for detection of infectious agents, such as influenza viruses, is that of nucleic acid sequence variation. DNA probes, 70-nt long, some including the nucleotide analog deoxyribose-Inosine (dInosine), were analyzed for hybridization tolerance to different amounts and distributions of mismatching bases, e.g. synonymous mutations, in target DNA. Microsphere-linked 70-mer probes were hybridized in 3M TMAC buffer to biotinylated single-stranded (ss) DNA for subsequent analysis in a Luminex® system. When mismatches interrupted contiguous matching stretches of 6 nt or longer, it had a strong impact on hybridization. Contiguous matching stretches are more important than the same number of matching nucleotides separated by mismatches into several regions. dInosine, but not 5-nitroindole, substitutions at mismatching positions stabilized hybridization remarkably well, comparable to N (4-fold) wobbles in the same positions. In contrast to shorter probes, 70-nt probes with judiciously placed dInosine substitutions and/or wobble positions were remarkably mismatch tolerant, with preserved specificity. An algorithm, NucZip, was constructed to model the nucleation and zipping phases of hybridization, integrating both local and distant binding contributions. It predicted hybridization more exactly than previous algorithms, and has the potential to guide the design of variation-tolerant yet specific probes.
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Multiplex quantitative PCR for detection of lower respiratory tract infection and meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis.
BMC Microbiol.
PUBLISHED: 06-07-2010
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Streptococcus pneumoniae and Haemophilus influenzae cause pneumonia and as Neisseria meningitidis they are important agents of meningitis. Although several PCR methods have been described for these bacteria the specificity is an underestimated problem. Here we present a quantitative multiplex real-time PCR (qmPCR) for detection of S. pneumoniae (9802 gene fragment), H. influenzae (omp P6 gene) and N. meningitidis (ctrA gene). The method was evaluated on bronchoalveolar lavage (BAL) samples from 156 adults with lower respiratory tract infection (LRTI) and 31 controls, and on 87 cerebrospinal fluid (CSF) samples from meningitis patients.
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Prevalence and phylogeny of coronaviruses in wild birds from the Bering Strait area (Beringia).
PLoS ONE
PUBLISHED: 04-20-2010
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Coronaviruses (CoVs) can cause mild to severe disease in humans and animals, their host range and environmental spread seem to have been largely underestimated, and they are currently being investigated for their potential medical relevance. Infectious bronchitis virus (IBV) belongs to gamma-coronaviruses and causes a costly respiratory viral disease in chickens. The role of wild birds in the epidemiology of IBV is poorly understood. In the present study, we examined 1,002 cloacal and faecal samples collected from 26 wild bird species in the Beringia area for the presence of CoVs, and then we performed statistical and phylogenetic analyses. We detected diverse CoVs by RT-PCR in wild birds in the Beringia area. Sequence analysis showed that the detected viruses are gamma-coronaviruses related to IBV. These findings suggest that wild birds are able to carry gamma-coronaviruses asymptomatically. We concluded that CoVs are widespread among wild birds in Beringia, and their geographic spread and frequency is higher than previously realised. Thus, Avian CoV can be efficiently disseminated over large distances and could be a genetic reservoir for future emerging pathogenic CoVs. Considering the great animal health and economic impact of IBV as well as the recent emergence of novel coronaviruses such as SARS-coronavirus, it is important to investigate the role of wildlife reservoirs in CoV infection biology and epidemiology.
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A microsphere-based immunoassay for rapid and sensitive detection of bovine viral diarrhoea virus antibodies.
J. Virol. Methods
PUBLISHED: 03-30-2010
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This study describes a novel blocking microsphere-based immunoassay for highly sensitive and specific detection of antibodies against bovine viral diarrhoea virus (BVDV). The intra- and inter-assay variability are 4.9% and less than 7%, respectively, and variability of bead conjugations is less than 6.6%. The diagnostic performance of the assay was evaluated by testing a total of 509 serum samples. Based on a negative/positive cut-off value of 30.3%, the assay has a sensitivity of 99.4% and a specificity of 98.3% relative to ELISA. The new microsphere immunoassay provides an alternative to conventional ELISA systems and can be used for high-throughput screening in the BVD control programmes.
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Arsenic trioxide influences viral replication in target organs of coxsackievirus B3-infected mice.
Microbes Infect.
PUBLISHED: 03-18-2010
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New antiviral agents are urgently needed. Based on in vitro studies, arsenic trioxide (As?O?) seems to affect viral replication, although this has been studied only marginally in vivo. In this study the replication of coxsackievirus B3 (CVB3) was studied in Balb/c mice administered 1 mg As?O?/kg bw once daily during 7 days of infection and in Vero cells exposed for 3 or 5 days to 0.4, 2 or 4 ?M As?O?. Viral RNA was measured by reverse transcription PCR (RT-PCR) (in vitro and in vivo) and arsenic concentration was measured by inductively coupled plasma-mass spectrometry (ICP-MS) (in vivo). In vivo, As?O? decreased viral RNA in the brain on days 3 (by 81%; p < 0.05) and 7 (by 97%; p < 0.01) and in the pancreas on day 7 (by 75%; p < 0.05), two of the target organs of this infection. The results were confirmed in vitro, where As?O? dose-dependently reduced viral RNA, with the effect being more pronounced in the surrounding culture medium than inside the infected cells, indicating an impaired virion release. Thus, As?O? reduced CVB3 replication both in vitro and in vivo, indicating that As?O? is a viable option in the pursuit of new therapeutic agents against viral infections.
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A novel combination of TaqMan RT-PCR and a suspension microarray assay for the detection and species identification of pestiviruses.
Vet. Microbiol.
PUBLISHED: 09-30-2009
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The genus pestivirus contains four recognized species: classical swine fever virus, border disease virus, bovine viral diarrhoea virus types 1 and 2. All are economically important and globally distributed but classical swine fever is the most serious, concerning losses and control measures. It affects both domestic pigs and wild boars. Outbreaks of this disease in domestic pigs call for the most serious measures of disease control, including a stamping out policy in Europe. Since all the members of the pestivirus genus can infect swine, differential diagnosis using traditional methods poses some problems. Antibody tests may lack specificity due to cross-reactions, antigen capture ELISAs may have low sensitivity, and virus isolation may take several days or even longer time to complete. PCR-based tests overcome these problems for the most part, but in general lack the multiplexing capability to detect and differentiate all the pestiviruses simultaneously. The assay platform described here addresses all of these issues by combining the advantages of real-time PCR with the multiplexing capability of microarray technology. The platform includes a TaqMan real-time PCR designed for the universal detection of pestiviruses and a microarray assay that can use the amplicons produced in the real-time PCR to identify the specific pestivirus.
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Broadly targeted triplex real-time PCR detection of influenza A, B and C viruses based on the nucleoprotein gene and a novel "MegaBeacon" probe strategy.
J. Virol. Methods
PUBLISHED: 09-19-2009
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A PCR assay that covers animal and human influenza A, B and C viruses, i.e., most of Orthomyxoviridae, is needed. Influenza types are distinguished based on differences in the nucleoprotein (NP) present in the virus. Conserved NP regions were therefore used to design a TaqMan-based triplex reverse transcription real-time PCR method. Variability of influenza A within the probe target region mandated the development of a novel molecular beacon, the "Mega" molecular beacon (MegaBeacon; MegB), for the detection of influenza A with this method. MegaBeacon is a mismatch-tolerant molecular beacon that is also a TaqMan probe. The triplex method (3QPCR-MegB) was evaluated with influenza A isolates covering 18 HxNx combinations, two influenza B isolates, and five Japanese influenza C isolates, as well as influenza A, B and C synthetic DNA targets. One to ten viral RNA and cDNA genome equivalents were detected per PCR reaction for influenza A, B and C. Seventy-one human nasopharyngeal aspirates from respiratory infections yielded 30 influenza A, 11 influenza B and 0 influenza C with 3QPCR-MegB, where immunofluorescence (IF) found 28 influenza A and 10 influenza B. 3QPCR-MegB was more mismatch-tolerant than a variant PCR with an influenza A TaqMan probe (3QPCR) and is a sensitive and rational method to detect influenza viruses of animal and human origin. MegaBeacon probes hold promise for variable target nucleic acids.
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RetroTector online, a rational tool for analysis of retroviral elements in small and medium size vertebrate genomic sequences.
BMC Bioinformatics
PUBLISHED: 06-16-2009
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The rapid accumulation of genomic information in databases necessitates rapid and specific algorithms for extracting biologically meaningful information. More or less complete retroviral sequences, also called proviral or endogenous retroviral sequences; ERVs, constitutes at least 5% of vertebrate genomes. After infecting the host, these retroviruses have integrated in germ line cells, and have then been carried in genomes for at least several 100 million years. A better understanding of structure and function of these sequences can have profound biological and medical consequences.
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Annotation and visualization of endogenous retroviral sequences using the Distributed Annotation System (DAS) and eBioX.
BMC Bioinformatics
PUBLISHED: 06-16-2009
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The Distributed Annotation System (DAS) is a widely used network protocol for sharing biological information. The distributed aspects of the protocol enable the use of various reference and annotation servers for connecting biological sequence data to pertinent annotations in order to depict an integrated view of the data for the final user.
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Multiplex and quantifiable detection of nucleic acid from pathogenic fungi using padlock probes, generic real time PCR and specific suspension array readout.
J. Microbiol. Methods
PUBLISHED: 05-21-2009
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A new concept for multiplex detection and quantification of microbes is here demonstrated on a range of infectious fungal species. Padlock probe methodology in conjunction with qPCR and Luminex technology was used for simultaneous detection of ten fungal species in one single experiment. By combining the multiplexing properties of padlock probes and Luminex detection with the well established quantitative characteristics of qPCR, quantitative microbe detection was done in 10-plex mode. A padlock probe is an oligonucleotide that via a ligation reaction forms circular DNA when hybridizing to specific target DNA. The region of the padlock probe that does not participate in target DNA hybridization contains generic primer sequences for amplification and a tag sequence for Luminex detection. This was the fundament for well performing multiplexing. Circularized padlock probes were initially amplified by rolling circle amplification (RCA), followed by a SybrGreen real time PCR which allowed an additive quantitative assessment of target DNA in the sample. Detection and quantification of amplified padlock probes were then done on color coded Luminex microspheres carrying anti-tag sequences. A novel technique, using labeled oligonucleotides to prevent reannealing of amplimers by covering the flanks of the address sequence, improved the signal to noise ratio in the detection step considerably. The method correctly detected fungi in a variety of clinical samples and offered quantitative information on fungal nucleic acid.
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Classification and nomenclature of endogenous retroviral sequences (ERVs): problems and recommendations.
Gene
PUBLISHED: 05-03-2009
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The genomes of many species are crowded with repetitive mobile sequences. In the case of endogenous retroviruses (ERVs) there is, for various reasons, considerable confusion regarding names assigned to families/groups of ERVs as well as individual ERV loci. Human ERVs have been studied in greater detail, and naming of HERVs in the scientific literature is somewhat confusing not just to the outsider. Without guidelines, confusion for ERVs in other species will also probably increase if those ERVs are studied in greater detail. Based on previous experience, this review highlights some of the problems when naming and classifying ERVs, and provides some guidance for detecting and characterizing ERV sequences. Because of the close relationship between ERVs and exogenous retroviruses (XRVs) it is reasonable to reconcile their classification with that of XRVs. We here argue that classification should be based on a combination of similarity, structural features, (inferred) function, and previous nomenclature. Because the RepBase system is widely employed in genome annotation, RepBase designations should be considered in further taxonomic efforts. To lay a foundation for a phylogenetically based taxonomy, further analyses of ERVs in many hosts are needed. A dedicated, permanent, international consortium would best be suited to integrate and communicate our current and future knowledge on repetitive, mobile elements in general to the scientific community.
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The phylogeny of orthoretroviral long terminal repeats (LTRs).
Gene
PUBLISHED: 04-29-2009
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LTRs are sequence elements in retroviruses and retrotransposons which are difficult to align due to their variability. One way of handling such cases is to use Hidden Markov Models (HMMs). In this work HMMs of LTRs were constructed for three groups of orthoretroviruses: the betaretroviruslike human MMTV-like (HML) endogenous retroviruses, the lentiviruses, including HIV, and gammaretroviruslike human endogenous retroviruses (HERVs). The HMM-generated LTR alignments and the phylogenetic trees constructed from them were compared with trees based on alignments of the pol gene at the nucleic acid level. The majority of branches in the LTR and pol based trees had the same order for the three retroviral genera, showing that HMM methods are successful in aligning and constructing phylogenies of LTRs. The HML LTR tree deviated somewhat from the pol tree for the groups HML3, HML7 and HML6. Among the gammaretroviruslike proviruses, the exogenous Mouse Leukemia Virus (MLV) was highly related to HERV-T in the pol based tree, but not in the LTR based tree. Aside from these differences, the similarity between the trees indicates that LTRs and pol coevolved in a largely monophyletic way.
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Broadly targeted multiprobe QPCR for detection of coronaviruses: Coronavirus is common among mallard ducks (Anas platyrhynchos).
J. Virol. Methods
PUBLISHED: 04-17-2009
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Coronaviruses (CoVs) can cause trivial or fatal disease in humans and in animals. Detection methods for a wide range of CoVs are needed, to understand viral evolution, host range, transmission and maintenance in reservoirs. A new concept, "Multiprobe QPCR", which uses a balanced mixture of competing discrete non- or moderately degenerated nuclease degradable (TaqMan) probes was employed. It provides a broadly targeted and rational single tube real-time reverse transcription PCR ("NQPCR") for the generic detection and discovery of CoV. Degenerate primers, previously published, and the new probes, were from a conserved stretch of open reading frame 1b, encoding the replicase. This multiprobe design reduced the degree of probe degeneration, which otherwise decreases the sensitivity, and allowed a preliminary classification of the amplified sequence directly from the QPCR trace. The split probe strategy allowed detection of down to 10 viral nucleic acid equivalents of CoV from all known CoV groups. Evaluation was with reference CoV strains, synthetic targets, human respiratory samples and avian fecal samples. Infectious-Bronchitis-Virus (IBV)-related variants were found in 7 of 35 sample pools, from 100 wild mallards (Anas platyrhynchos). Ducks may spread and harbour CoVs. NQPCR can detect a wide range of CoVs, as illustrated for humans and ducks.
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Detection of Haemophilus influenzae in respiratory secretions from pneumonia patients by quantitative real-time polymerase chain reaction.
Diagn. Microbiol. Infect. Dis.
PUBLISHED: 03-26-2009
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A quantitative real-time polymerase chain reaction (PCR) based on the omp P6 gene was developed to detect Haemophilus influenzae. Its specificity was determined by analysis of 29 strains of 11 different Haemophilus spp. and was compared with PCR assays having other target genes: rnpB, 16S rRNA, and bexA. The method was evaluated on nasopharyngeal aspirates from 166 adult patients with community-acquired pneumonia. When 10(4) DNA copies/mL was used as cutoff limit for the method, P6 PCR had a sensitivity of 97.5% and a specificity of 96.0% compared with the culture. Of 20 culture-negative but P6 PCR-positive cases, 18 were confirmed by fucK PCR as H. influenzae. Five (5.9%) of 84 nasopharyngeal aspirates from adult controls tested PCR positive. We conclude that the P6 real-time PCR is both sensitive and specific for identification of H. influenzae in respiratory secretions. Quantification facilitates discrimination between disease-causing H. influenzae strains and commensal colonization.
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Polybrominated diphenyl ether exposure suppresses cytokines important in the defence to coxsackievirus B3 infection in mice.
Toxicol. Lett.
PUBLISHED: 03-18-2009
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Environmental pollutants can adversely affect the immune system. The host defence during infection depends on cytokine signalling and proper function of immune cells. However, no studies have addressed how polybrominated diphenyl ethers (PBDEs) affect cytokine responses. We investigated the combined effects in Balb/c mice of human coxsackievirus B3 (CVB3) infection and exposure to PBDEs (BDE-99 or Bromkal mixture) on 21 serum cytokines. The mice were infected (i.p.) on day 0, orally treated with BDE-99 or Bromkal on day 1 (20mg/kg bw) and put to death on day 3. CVB3 was quantitatively measured in the liver and pancreas by RT-PCR. The Luminex 200 multi-analyte system was used for cytokine analysis. High numbers of viral copies were found in the liver and pancreas. Infection increased TNF-alpha, IL-6, MCP-1, IL-12p40, KC and RANTES levels. Notably, PBDE-exposure resulted in a marked decrease, or even lack, of IL-13, MIP-1beta, RANTES, IFN-gamma and KC levels in non-infected mice. However, the effects of PBDE-exposure on cytokines did not affect viral replication during early CVB3 infection. In conclusion, PBDEs causes a selective block in immune signalling pathways but the consequences of this need to be further studied in different host resistance models of infection.
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Development of a magnetic bead microarray for simultaneous and simple detection of four pestiviruses.
J. Virol. Methods
PUBLISHED: 03-18-2009
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This study reports a novel method for the rapid detection and identification of the four recognized species in the pestivirus genus of the Flaviviridae family, i.e. classical swine fever virus (CSFV), border disease virus (BDV), bovine viral diarrhoea virus type 1 (BVDV1) and type 2 (BVDV2). The analysis of pestivirus PCR products was performed on microarrays by means of magnetic bead detection. The process utilizes an oligonucleotide array, onto which 5 biotinylated PCR products were hybridized, followed by visualization with streptavidin-coated magnetic particles by the naked eye, microscope or biochip reader. The assay was tested on a collection of pestiviruses that included all four species and allowed a specific and sensitive detection. Sensitivity was compared with other post-PCR detection methods, namely gel electrophoresis and suspension microarray. The results indicate that due to its high sensitivity, specificity and simple detection procedure, the magnetic bead assay provides a powerful tool for detection and identification of viral pathogens. Considering the simplicity of the assay, the protocols for hybridization and magnetic bead detection offer an emerging application for molecular diagnoses in virology that is amenable for use in a modestly equipped laboratory.
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Evolutionary conservation of orthoretroviral long terminal repeats (LTRs) and ab initio detection of single LTRs in genomic data.
PLoS ONE
PUBLISHED: 03-10-2009
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Retroviral LTRs, paired or single, influence the transcription of both retroviral and non-retroviral genomic sequences. Vertebrate genomes contain many thousand endogenous retroviruses (ERVs) and their LTRs. Single LTRs are difficult to detect from genomic sequences without recourse to repetitiveness or presence in a proviral structure. Understanding of LTR structure increases understanding of LTR function, and of functional genomics. Here we develop models of orthoretroviral LTRs useful for detection in genomes and for structural analysis.
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Sequential changes in serum cytokines reflect viral RNA kinetics in target organs of a coxsackievirus B infection in mice.
J. Clin. Immunol.
PUBLISHED: 02-23-2009
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The pattern of cytokine responses related to viral replication during the course of the common human coxsackievirus B3 (CVB3) infection is not known.
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Analyses of pig genomes provide insight into porcine demography and evolution.
Martien A M Groenen, Alan L Archibald, Hirohide Uenishi, Christopher K Tuggle, Yasuhiro Takeuchi, Max F Rothschild, Claire Rogel-Gaillard, Chankyu Park, Denis Milan, Hendrik-Jan Megens, Shengting Li, Denis M Larkin, Heebal Kim, Laurent A F Frantz, Mario Caccamo, Hyeonju Ahn, Bronwen L Aken, Anna Anselmo, Christian Anthon, Loretta Auvil, Bouabid Badaoui, Craig W Beattie, Christian Bendixen, Daniel Berman, Frank Blecha, Jonas Blomberg, Lars Bolund, Mirte Bosse, Sara Botti, Zhan Bujie, Megan Bystrom, Boris Capitanu, Denise Carvalho-Silva, Patrick Chardon, Celine Chen, Ryan Cheng, Sang-Haeng Choi, William Chow, Richard C Clark, Christopher Clee, Richard P M A Crooijmans, Harry D Dawson, Patrice Dehais, Fioravante De Sapio, Bert Dibbits, Nizar Drou, Zhi-Qiang Du, Kellye Eversole, Joao Fadista, Susan Fairley, Thomas Faraut, Geoffrey J Faulkner, Katie E Fowler, Merete Fredholm, Eric Fritz, James G R Gilbert, Elisabetta Giuffra, Jan Gorodkin, Darren K Griffin, Jennifer L Harrow, Alexander Hayward, Kerstin Howe, Zhi-Liang Hu, Sean J Humphray, Toby Hunt, Henrik Hornshøj, Jin-Tae Jeon, Patric Jern, Matthew Jones, Jerzy Jurka, Hiroyuki Kanamori, Ronan Kapetanovic, Jaebum Kim, Jae-Hwan Kim, Kyu-Won Kim, Tae-Hun Kim, Greger Larson, Kyooyeol Lee, Kyung-Tai Lee, Richard Leggett, Harris A Lewin, Yingrui Li, Wansheng Liu, Jane E Loveland, Yao Lu, Joan K Lunney, Jian Ma, Ole Madsen, Katherine Mann, Lucy Matthews, Stuart McLaren, Takeya Morozumi, Michael P Murtaugh, Jitendra Narayan, Dinh Truong Nguyen, Peixiang Ni, Song-Jung Oh, Suneel Onteru, Frank Panitz, Eung-Woo Park, Hong-Seog Park, Géraldine Pascal, Yogesh Paudel, Miguel Pérez-Enciso, Ricardo Ramirez-Gonzalez, James M Reecy, Sandra Rodriguez-Zas, Gary A Rohrer, Lauretta Rund, Yongming Sang, Kyle Schachtschneider, Joshua G Schraiber, John Schwartz, Linda Scobie, Carol Scott, Stephen Searle, Bertrand Servin, Bruce R Southey, Göran Sperber, Peter Stadler, Jonathan V Sweedler, Hakim Tafer, Bo Thomsen, Rashmi Wali, Jian Wang, Jun Wang, Simon White, Xun Xu, Martine Yerle, Guojie Zhang, Jianguo Zhang, Jie Zhang, Shuhong Zhao, Jane Rogers, Carol Churcher, Lawrence B Schook.
Nature
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For 10,000?years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ?1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model.
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Unexpected diversity and expression of avian endogenous retroviruses.
MBio
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Endogenous retroviruses (ERVs) were identified and characterized in three avian genomes to gain insight into early retroviral evolution. Using the computer program RetroTector to detect relatively intact ERVs, we identified 500 ERVs in the chicken genome, 150 in the turkey genome, and 1,200 in the zebra finch genome. Previous studies suggested that endogenous alpharetroviruses were present in chicken genomes. In this analysis, a small number of alpharetroviruses were seen in the chicken and turkey genomes; however, these were greatly outnumbered by beta-like, gamma-like, and alphabeta proviruses. While the avian ERVs belonged to the same major groups as mammalian ERVs, they were more heterogeneous. In particular, the beta-like viruses revealed an evolutionary continuum with the gradual acquisition and loss of betaretroviral markers and a transition from beta to alphabeta and then to alpharetroviruses. Thus, it appears that birds may resemble a melting pot for early ERV evolution. Many of the ERVs were integrated in clusters on chromosomes, often near centromeres. About 25% of the chicken ERVs were in or near cellular transcription units; this is nearly random. The majority of these integrations were in the sense orientation in introns. A higher-than-random number of integrations were >100 kb from the nearest gene. Deep-sequencing studies of chicken embryo fibroblasts revealed that about 20% of the 500 ERVs were transcribed and translated. A subset of these were also transcribed in vivo in chickens, showing tissue-specific patterns of expression. IMPORTANCE Studies of avian endogenous retroviruses (ERVs) have given us a glimpse of an earlier retroviral world. Three different classes of ERVs were observed with many features of mammalian retroviruses, as well as some important differences. Many avian ERVs were transcribed and translated.
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Variation-tolerant capture and multiplex detection of nucleic acids: application to detection of microbes.
J. Clin. Microbiol.
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In contrast to ordinary PCRs, which have a limited multiplex capacity and often return false-negative results due to target variation or inhibition, our new detection strategy, VOCMA (variation-tolerant capture multiplex assay), allows variation-tolerant, target-specific capture and detection of many nucleic acids in one test. Here we demonstrate the use of a single-tube, dual-step amplification strategy that overcomes the usual limitations of PCR multiplexing, allowing at least a 22-plex format with retained sensitivity. Variation tolerance was achieved using long primers and probes designed to withstand variation at known sites and a judicious mix of degeneration and universal bases. We tested VOCMA in situations where enrichment from a large sample volume with high sensitivity and multiplexity is important (sepsis; streptococci, enterococci, and staphylococci, several enterobacteria, candida, and the most important antibiotic resistance genes) and where variation tolerance and high multiplexity is important (gastroenteritis; astrovirus, adenovirus, rotavirus, norovirus genogroups I and II, and sapovirus, as well as enteroviruses, which are not associated with gastroenteritis). Detection sensitivities of 10 to 1,000 copies per reaction were achieved for many targets. VOCMA is a highly multiplex, variation-tolerant, general purpose nucleic acid detection concept. It is a specific and sensitive method for simultaneous detection of nucleic acids from viruses, bacteria, fungi, and protozoa, as well as host nucleic acid, in the same test. It can be run on an ordinary PCR and a Luminex machine and is suitable for both clinical diagnoses and microbial surveillance.
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No evidence for xenotropic murine leukemia-related virus infection in Sweden using internally controlled multiepitope suspension array serology.
Clin. Vaccine Immunol.
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Many syndromes have a large number of differential diagnoses, a situation which calls for multiplex diagnostic systems. Myalgic encephalomyelitis (ME), also named chronic fatigue syndrome (CFS), is a common disease of unknown etiology. A mouse retrovirus, xenotropic murine leukemia-related virus (XMRV), was found in ME/CFS patients and blood donors, but this was not corroborated. However, the paucity of serological investigations on XMRV in humans prompted us to develop a serological assay which cover many aspects of XMRV antigenicity. It is a novel suspension array method, using a multiplex IgG assay with nine recombinant proteins from the env and gag genes of XMRV and 38 peptides based on known epitopes of vertebrate gammaretroviruses. IgG antibodies were sought in 520 blood donors and 85 ME/CFS patients and in positive- and negative-control sera from animals. We found no differences in seroreactivity between blood donors and ME/CFS patients for any of the antigens. This did not support an association between ME/CFS and XMRV infection. The multiplex serological system had several advantages: (i) biotinylated protein G allowed us to run both human and animal sera, which is essential because of a lack of XMRV-positive humans; (ii) a novel quality control was a pan-peptide positive-control rabbit serum; and (iii) synthetic XMRV Gag peptides with degenerate positions covering most of the variation of murine leukemia-like viruses did not give higher background than nondegenerate analogs. The principle may be used for creation of variant tolerant peptide serologies. Thus, our system allows rational large-scale serological assays with built-in quality control.
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Detection of subgenomic mRNA of feline coronavirus by real-time polymerase chain reaction based on primer-probe energy transfer (P-sg-QPCR).
J. Virol. Methods
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Feline infectious peritonitis is one of the most severe devastating diseases of the Felidae. Upon the appearance of clinical signs, a cure for the infected animal is impossible. Therefore rapid and proper diagnosis for both the presence of the causative agent, feline coronavirus (FCoV) and the manifestation of feline infectious peritonitis is of paramount importance. In the present work, a novel real-time RT-PCR method is described which is able to detect FCoV and to determine simultaneously the quantity of the viral RNA. The new assay combines the M gene subgenomic messenger RNA (sg-mRNA) detection and the quantitation of the genome copies of FCoV. In order to detect the broadest spectrum of potential FCoV variants and to achieve the most accurate results in the detection ability the new assay is applying the primer-probe energy transfer (PriProET) principle. This technology was chosen since PriProET is very robust to tolerate the nucleotide substitutions in the target area. Therefore, this technology provides a very broad-range system, which is able to detect simultaneously many variants of the virus(es) even if the target genomic regions show large scale of variations. The detection specificity of the new assay was proven by positive amplification from a set of nine different FCoV strains and negative from the tested non-coronaviral targets. Examination of faecal samples of healthy young cats, organ samples of perished animals, which suffered from feline infectious peritonitis, and cat leukocytes from uncertain clinical cases were also subjected to the assay. The sensitivity of the P-sg-QPCR method was high, since as few as 10 genome copies of FCoV were detected. The quantitative sg-mRNA detection method revealed more than 10-50,000 times increase of the M gene sg-mRNA in organ materials of feline infectious peritonitis cases, compared to those of the enteric FCoV variants present in the faeces of normal, healthy cats. These results indicate the applicability of the new P-sg-QPCR test as a powerful novel tool for the better detection and quantitation of FCoV and for the improved diagnosis of feline infectious peritonitis, this important disease of the Felidae, causing serious losses in the cat populations at a global scale.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.