共查询到20条相似文献,搜索用时 500 毫秒
1.
Sachsenröder J Twardziok S Hammerl JA Janczyk P Wrede P Hertwig S Johne R 《PloS one》2012,7(4):e34631
Background
Animal faeces comprise a community of many different microorganisms including bacteria and viruses. Only scarce information is available about the diversity of viruses present in the faeces of pigs. Here we describe a protocol, which was optimized for the purification of the total fraction of viral particles from pig faeces. The genomes of the purified DNA and RNA viruses were simultaneously amplified by PCR and subjected to deep sequencing followed by bioinformatic analyses. The efficiency of the method was monitored using a process control consisting of three bacteriophages (T4, M13 and MS2) with different morphology and genome types. Defined amounts of the bacteriophages were added to the sample and their abundance was assessed by quantitative PCR during the preparation procedure.Results
The procedure was applied to a pooled faecal sample of five pigs. From this sample, 69,613 sequence reads were generated. All of the added bacteriophages were identified by sequence analysis of the reads. In total, 7.7% of the reads showed significant sequence identities with published viral sequences. They mainly originated from bacteriophages (73.9%) and mammalian viruses (23.9%); 0.8% of the sequences showed identities to plant viruses. The most abundant detected porcine viruses were kobuvirus, rotavirus C, astrovirus, enterovirus B, sapovirus and picobirnavirus. In addition, sequences with identities to the chimpanzee stool-associated circular ssDNA virus were identified. Whole genome analysis indicates that this virus, tentatively designated as pig stool-associated circular ssDNA virus (PigSCV), represents a novel pig virus.Conclusion
The established protocol enables the simultaneous detection of DNA and RNA viruses in pig faeces including the identification of so far unknown viruses. It may be applied in studies investigating aetiology, epidemiology and ecology of diseases. The implemented process control serves as quality control, ensures comparability of the method and may be used for further method optimization. 相似文献2.
Background
MicroRNAs (miRNAs) represent new and potentially informative diagnostic targets for disease detection and prognosis. However, little work exists documenting the effect of TRIzol, a common viral inactivation and nucleic acid extraction reagent, on miRNA purification. Here, we developed an optimized protocol for miRNA extraction from plasma samples by evaluating five different RNA extraction kits, TRIzol phase separation, purification additives, and initial plasma sample volume. This method was then used for downstream profiling of plasma miRNAs found in archived samples from one nonhuman primate (NHP) experimentally challenged with Ebola virus by the aerosol route.Results
Comparison of real-time RT-PCR results for spiked-in and endogenous miRNA sequences determined extraction efficiencies from five different RNA purification kits. These experiments showed that 50 μL plasma processed using the QIAGEN miRNeasy Mini Kit with 5 μg of glycogen as a co-precipitant yielded the highest recovery of endogenous miRNAs. Using this optimized protocol, miRNAs from archived plasma samples of one rhesus macaque challenged with aerosolized Ebola virus was profiled using a targeted real-time PCR array. A total of 519 of the 752 unique miRNAs assayed were present in the plasma samples at day 0 and day 7 (time of death) post-exposure. Statistical analyses revealed 25 sequences significantly up- or down-regulated between day 0 and day 7 post infection, validating the utility of the extraction method for plasma miRNA profiling.Conclusions
This study contributes to the knowledgebase of circulating miRNA extraction methods and expands on the potential applications of cell-free miRNA profiling for diagnostics and pathogenesis studies. Specifically, we optimized an extraction protocol for miRNAs from TRIzol-inactivated plasma samples that can be used for highly pathogenic viruses.Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1299-5) contains supplementary material, which is available to authorized users. 相似文献3.
Jan Felix Drexler Victor Max Corman Florian Gloza-Rausch Antje Seebens Augustina Annan Anne Ipsen Thomas Kruppa Marcel A. Müller Elisabeth K. V. Kalko Yaw Adu-Sarkodie Samuel Oppong Christian Drosten 《PloS one》2009,4(7)
Background
Henipaviruses (Hendra and Nipah virus) are highly pathogenic members of the family Paramyxoviridae. Fruit-eating bats of the Pteropus genus have been suggested as their natural reservoir. Human Henipavirus infections have been reported in a region extending from Australia via Malaysia into Bangladesh, compatible with the geographic range of Pteropus. These bats do not occur in continental Africa, but a whole range of other fruit bats is encountered. One of the most abundant is Eidolon helvum, the African Straw-coloured fruit bat.Methodology/Principal Findings
Feces from E. helvum roosting in an urban setting in Kumasi/Ghana were tested for Henipavirus RNA. Sequences of three novel viruses in phylogenetic relationship to known Henipaviruses were detected. Virus RNA concentrations in feces were low.Conclusions/Significance
The finding of novel putative Henipaviruses outside Australia and Asia contributes a significant extension of the region of potential endemicity of one of the most pathogenic virus genera known in humans. 相似文献4.
Background
Autistic spectrum disorder (ASD) is characterized by impaired language, communication and social skills, as well as by repetitive and stereotypic patterns of behavior. Many autistic subjects display a dysregulation of the immune system which is compatible with an unresolved viral infection with prenatal onset, potentially due to vertical viral transmission. Recently, the xenotropic murine leukemia virus-related virus (XMRV) has been implicated in chronic fatigue syndrome (CFS) and in prostate cancer by several, though not all studies.Methodology/Principal Findings
We assessed whether XMRV or other murine leukemia virus (MLV)-related viruses are involved in autistic disorder. Using nested PCR targeted to gag genomic sequences, we screened DNA samples from: (i) peripheral blood of 102 ASD patients and 97 controls, (ii) post-mortem brain samples of 20 ASD patients and 17 sex- and age-matched controls, (iii) semen samples of 11 fathers of ASD children, 25 infertile individuals and 7 fertile controls. No XMRV gag DNA sequences were detected, whereas peripheral blood samples of 3/97 (3.1%) controls were positive for MLV.Conclusions|Significance
No MLV-related virus was detected in blood, brain, and semen samples of ASD patients or fathers. Hence infection with XMRV or other MLV-related viruses is unlikely to contribute to autism pathogenesis. 相似文献5.
Background
The current spread of pandemic influenza A(H1N1)v virus necessitates an intensified surveillance of influenza virus infections worldwide. So far, in many laboratories routine diagnostics were limited to generic influenza virus detection only. To provide interested laboratories with real-time PCR assays for type and subtype identification, we present a bundle of PCR assays with which any human influenza A and B virus can be easily identified, including assays for the detection of the pandemic A(H1N1)v virus.Principal Findings
The assays show optimal performance characteristics in their validation on plasmids containing the respective assay target sequences. All assays have furthermore been applied to several thousand clinical samples since 2007 (assays for seasonal influenza) and April 2009 (pandemic influenza assays), respectively, and showed excellent results also on clinical material.Conclusions
We consider the presented assays to be well suited for the detection and subtyping of circulating influenza viruses. 相似文献6.
Tang S Zhao J Haleyur Giri Setty MK Devadas K Gaddam D Viswanath R Wood O Zhang P Hewlett IK 《PloS one》2011,6(11):e27391
Background
Preliminary studies in chronic fatigue syndrome (CFS) patients and XMRV infected animals demonstrated plasma viremia and infection of blood cells with XMRV, indicating the potential risk for transfusion transmission. XMRV and MLV-related virus gene sequences have also been detected in 4–6% of healthy individuals including blood donors in the U.S. These results imply that millions of persons in the U.S. may be carrying the nucleic acid sequences of XMRV and/or MLV-related viruses, which is a serious public health and blood safety concern.Methodology/Principal Findings
To gain evidence of XMRV or MLV-related virus infection in the U.S. blood donors, 110 plasma samples and 71 PBMC samples from blood donors at the NIH blood bank were screened for XMRV and MLV-related virus infection. We employed highly sensitive assays, including nested PCR and real-time PCR, as well as co-culture of plasma with highly sensitive indicator DERSE cells. Using these assays, none of the samples were positive for XMRV or MLV-related virus.Conclusions/Significance
Our results are consistent with those from several other studies, and demonstrate the absence of XMRV or MLV-related viruses in the U.S. blood donors that we studied. 相似文献7.
Rachel Brower-Sinning Diana Zhong Misty Good Brian Firek Robyn Baker Chhinder P. Sodhi David J. Hackam Michael J. Morowitz 《PloS one》2014,9(9)
Background
Previous studies of infant fecal samples have failed to clarify the role of gut bacteria in the pathogenesis of NEC. We sought to characterize bacterial communities within intestinal tissue resected from infants with and without NEC.Methods
26 intestinal samples were resected from 19 infants, including 16 NEC samples and 10 non-NEC samples. Bacterial 16S rRNA gene sequences were amplified and sequenced. Analysis allowed for taxonomic identification, and quantitative PCR was used to quantify the bacterial load within samples.Results
NEC samples generally contained an increased total burden of bacteria. NEC and non-NEC sample sets were both marked by high inter-individual variability and an abundance of opportunistic pathogens. There was no statistically significant distinction between the composition of NEC and non-NEC microbial communities. K-means clustering enabled us to identify several stable clusters, including clusters of NEC and midgut volvulus samples enriched with Clostridium and Bacteroides. Another cluster containing both NEC and non-NEC samples was marked by an abundance of Enterobacteriaceae and decreased diversity among NEC samples.Conclusions
The results indicate that NEC is a disease without a uniform pattern of microbial colonization, but that NEC is associated with an abundance of strict anaerobes and a decrease in community diversity. 相似文献8.
Adam Schayowitz Greg Bertenshaw Emiko Jeffries Timothy Schatz James Cotton Jessie Villanueva Meenhard Herlyn Clemens Krepler Adina Vultur Wei Xu Gordon H. Yu Lynn Schuchter Douglas P. Clark 《PloS one》2012,7(12)
Aims
This proof-of-concept study was designed to determine if functional, pharmacodynamic profiles relevant to targeted therapy could be derived from live human melanoma samples using a novel automated platform.Methods
A series of 13 melanoma cell lines was briefly exposed to a BRAF inhibitor (PLX-4720) on a platform employing automated fluidics for sample processing. Levels of the phosphoprotein p-ERK in the mitogen-activated protein kinase (MAPK) pathway from treated and untreated sample aliquots were determined using a bead-based immunoassay. Comparison of these levels provided a determination of the pharmacodynamic effect of the drug on the MAPK pathway. A similar ex vivo analysis was performed on fine needle aspiration (FNA) biopsy samples from four murine xenograft models of metastatic melanoma, as well as 12 FNA samples from patients with metastatic melanoma.Results
Melanoma cell lines with known sensitivity to BRAF inhibitors displayed marked suppression of the MAPK pathway in this system, while most BRAF inhibitor-resistant cell lines showed intact MAPK pathway activity despite exposure to a BRAF inhibitor (PLX-4720). FNA samples from melanoma xenografts showed comparable ex vivo MAPK activity as their respective cell lines in this system. FNA samples from patients with metastatic melanoma successfully yielded three categories of functional profiles including: MAPK pathway suppression; MAPK pathway reactivation; MAPK pathway stimulation. These profiles correlated with the anticipated MAPK activity, based on the known BRAF mutation status, as well as observed clinical responses to BRAF inhibitor therapy.Conclusion
Pharmacodynamic information regarding the ex vivo effect of BRAF inhibitors on the MAPK pathway in live human melanoma samples can be reproducibly determined using a novel automated platform. Such information may be useful in preclinical and clinical drug development, as well as predicting response to targeted therapy in individual patients. 相似文献9.
Michael A Quail Miriam Smith David Jackson Steven Leonard Thomas Skelly Harold P Swerdlow Yong Gu Peter Ellis 《BMC genomics》2014,15(1)
Background
A minor but significant fraction of samples subjected to next-generation sequencing methods are either mixed-up or cross-contaminated. These events can lead to false or inconclusive results. We have therefore developed SASI-Seq; a process whereby a set of uniquely barcoded DNA fragments are added to samples destined for sequencing. From the final sequencing data, one can verify that all the reads derive from the original sample(s) and not from contaminants or other samples.Results
By adding a mixture of three uniquely barcoded amplicons, of different sizes spanning the range of insert sizes one would normally use for Illumina sequencing, at a spike-in level of approximately 0.1%, we demonstrate that these fragments remain intimately associated with the sample. They can be detected following even the tightest size selection regimes or exome enrichment and can report the occurrence of sample mix-ups and cross-contamination.As a consequence of this work, we have designed a set of 384 eleven-base Illumina barcode sequences that are at least 5 changes apart from each other, allowing for single-error correction and very low levels of barcode misallocation due to sequencing error.Conclusion
SASI-Seq is a simple, inexpensive and flexible tool that enables sample assurance, allows deconvolution of sample mix-ups and reports levels of cross-contamination between samples throughout NGS workflows.Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-110) contains supplementary material, which is available to authorized users. 相似文献10.
Otto Erlwein Steve Kaye Myra O. McClure Jonathan Weber Gillian Wills David Collier Simon Wessely Anthony Cleare 《PloS one》2010,5(1)
Background
In October 2009 it was reported that 68 of 101 patients with chronic fatigue syndrome (CFS) in the US were infected with a novel gamma retrovirus, xenotropic murine leukaemia virus-related virus (XMRV), a virus previously linked to prostate cancer. This finding, if confirmed, would have a profound effect on the understanding and treatment of an incapacitating disease affecting millions worldwide. We have investigated CFS sufferers in the UK to determine if they are carriers of XMRV.Methodology
Patients in our CFS cohort had undergone medical screening to exclude detectable organic illness and met the CDC criteria for CFS. DNA extracted from blood samples of 186 CFS patients were screened for XMRV provirus and for the closely related murine leukaemia virus by nested PCR using specific oligonucleotide primers. To control for the integrity of the DNA, the cellular beta-globin gene was amplified. Negative controls (water) and a positive control (XMRV infectious molecular clone DNA) were included. While the beta-globin gene was amplified in all 186 samples, neither XMRV nor MLV sequences were detected.Conclusion
XMRV or MLV sequences were not amplified from DNA originating from CFS patients in the UK. Although we found no evidence that XMRV is associated with CFS in the UK, this may be a result of population differences between North America and Europe regarding the general prevalence of XMRV infection, and might also explain the fact that two US groups found XMRV in prostate cancer tissue, while two European studies did not. 相似文献11.
Background and Aims
Winter-flowering plants outside the tropics may experience a shortage of pollinator service, given that insect activity is largely limited by low temperature. Birds can be alternative pollinators for these plants, but experimental evidence for the pollination role of birds in winter-flowering plants is scarce.Methods
Pollinator visitation to the loquat, Eriobotrya japonica (Rosaceae), was observed across the flowering season from November to January for two years in central China. Self- and cross-hand pollination was conducted in the field to investigate self-compatibility and pollen limitation. In addition, inflorescences were covered by bird cages and nylon mesh nets to exclude birds and all animal pollinators, respectively, to investigate the pollination role of birds in seed production.Results
Self-fertilization in the loquat yielded few seeds. In early winter insect visit frequency was relatively higher, while in late winter insect pollinators were absent and two passerine birds (Pycnonotus sinensis and Zosterops japonicus) became the major floral visitors. However, seed-set of open-pollinated flowers did not differ between early and late winter. Exclusion of bird visitation greatly reduced seed-set, indicating that passerine birds were important pollinators for the loquat in late winter. The whitish perigynous flowers reward passerines with relatively large volumes of dilute nectar. Our observation on the loquat and other Rosaceae species suggested that perigyny might be related to bird pollination but the association needs further study.Conclusions
These findings suggest that floral traits and phenology would be favoured to attract bird pollinators in cold weather, in which insect activity is limited. 相似文献12.
Yu J Li H Tang Q Rayner S Han N Guo Z Liu H Adams J Fang W Tao X Wang S Liang G 《PLoS neglected tropical diseases》2012,6(5):e1640
Background and Objectives
Recent years have seen a rapid increase in the number of rabies cases in China and an expansion in the geographic distribution of the virus. In spite of the seriousness of the outbreak and increasing number of fatalities, little is known about the phylogeography of the disease in China. In this study, we report an analysis of a set of Nucleocapsid sequences consisting of samples collected through the trial Chinese National Surveillance System as well as publicly available sequences. This sequence set represents the most comprehensive dataset from China to date, comprising 210 sequences (including 57 new samples) from 15 provinces and covering all epidemic regions. Using this dataset we investigated genetic diversity, patterns of distribution, and evolutionary history.Results
Our analysis indicates that the rabies virus in China is primarily defined by two clades that exhibit distinct population subdivision and translocation patterns and that contributed to the epidemic in different ways. The younger clade originated around 1992 and has properties that closely match the observed spread of the recent epidemic. The older clade originated around 1960 and has a dispersion pattern that suggests it represents a strain associated with a previous outbreak that remained at low levels throughout the country and reemerged in the current epidemic.Conclusions
Our findings provide new insight into factors associated with the recent epidemic and are relevant to determining an effective policy for controlling the virus. 相似文献13.
Lei Yan Yan Gao Yong Zhang Michael Tildesley Liqi Liu Ye Zhang Leying Wen Wei Wang Xiaodan Li Ying Hu Tian Bai Min Wang Yuhong Zeng Dingming Wang Xianjun Wang Yu Lan Shiwen Wang Yuelong Shu 《PloS one》2012,7(9)
Background
During the 2009 pandemic influenza H1N1 (2009) virus (pH1N1) outbreak, school students were at an increased risk of infection by the pH1N1 virus. However, the estimation of the attack rate showed significant variability.Methods
Two school outbreaks were investigated in this study. A questionnaire was designed to collect information by interview. Throat samples were collected from all the subjects in this study 6 times and sero samples 3 times to confirm the infection and to determine viral shedding. Data analysis was performed using the software STATA 9.0.Findings
The attack rate of the pH1N1 outbreak was 58.3% for the primary school, and 52.9% for the middle school. The asymptomatic infection rates of the two schools were 35.8% and 37.6% respectively. Peak virus shedding occurred on the day of ARI symptoms onset, followed by a steady decrease over subsequent days (p = 0.026). No difference was found either in viral shedding or HI titer between the symptomatic and the asymptomatic infectious groups.Conclusions
School children were found to be at a high risk of infection by the novel virus. This may be because of a heightened risk of transmission owing to increased mixing at boarding school, or a lack of immunity owing to socio-economic status. We conclude that asymptomatically infectious cases may play an important role in transmission of the pH1N1 virus. 相似文献14.
Background
With an estimated 38 million people worldwide currently infected with human immunodeficiency virus (HIV), and an additional 4.1 million people becoming infected each year, it is important to understand how this virus mutates and develops resistance in order to design successful therapies.Methodology/Principal Findings
We report a novel experimental method for amplifying full-length HIV genomes without the use of sequence-specific primers for high throughput DNA sequencing, followed by assembly of full length viral genome sequences from the resulting large dataset. Illumina was chosen for sequencing due to its ability to provide greater coverage of the HIV genome compared to prior methods, allowing for more comprehensive characterization of the heterogeneity present in the HIV samples analyzed. Our novel amplification method in combination with Illumina sequencing was used to analyze two HIV populations: a homogenous HIV population based on the canonical NL4-3 strain and a heterogeneous viral population obtained from a HIV patient''s infected T cells. In addition, the resulting sequence was analyzed using a new computational approach to obtain a consensus sequence and several metrics of diversity.Significance
This study demonstrates how a lower bias amplification method in combination with next generation DNA sequencing provides in-depth, complete coverage of the HIV genome, enabling a stronger characterization of the quasispecies present in a clinically relevant HIV population as well as future study of how HIV mutates in response to a selective pressure. 相似文献15.
Prosser DJ Cui P Takekawa JY Tang M Hou Y Collins BM Yan B Hill NJ Li T Li Y Lei F Guo S Xing Z He Y Zhou Y Douglas DC Perry WM Newman SH 《PloS one》2011,6(3):e17622
Background
Qinghai Lake in central China has been at the center of debate on whether wild birds play a role in circulation of highly pathogenic avian influenza virus H5N1. In 2005, an unprecedented epizootic at Qinghai Lake killed more than 6000 migratory birds including over 3000 bar-headed geese (Anser indicus). H5N1 subsequently spread to Europe and Africa, and in following years has re-emerged in wild birds along the Central Asia flyway several times.Methodology/Principal Findings
To better understand the potential involvement of wild birds in the spread of H5N1, we studied the movements of bar-headed geese marked with GPS satellite transmitters at Qinghai Lake in relation to virus outbreaks and disease risk factors. We discovered a previously undocumented migratory pathway between Qinghai Lake and the Lhasa Valley of Tibet where 93% of the 29 marked geese overwintered. From 2003–2009, sixteen outbreaks in poultry or wild birds were confirmed on the Qinghai-Tibet Plateau, and the majority were located within the migratory pathway of the geese. Spatial and temporal concordance between goose movements and three potential H5N1 virus sources (poultry farms, a captive bar-headed goose facility, and H5N1 outbreak locations) indicated ample opportunities existed for virus spillover and infection of migratory geese on the wintering grounds. Their potential as a vector of H5N1 was supported by rapid migration movements of some geese and genetic relatedness of H5N1 virus isolated from geese in Tibet and Qinghai Lake.Conclusions/Significance
This is the first study to compare phylogenetics of the virus with spatial ecology of its host, and the combined results suggest that wild birds play a role in the spread of H5N1 in this region. However, the strength of the evidence would be improved with additional sequences from both poultry and wild birds on the Qinghai-Tibet Plateau where H5N1 has a clear stronghold. 相似文献16.
Kristin Fenton Silje Fismen Annica Hedberg Natalya Seredkina Chris Fenton Elin Synn?ve Mortensen Ole Petter Rekvig 《PloS one》2009,4(12)
Background
Lupus nephritis is characterized by deposition of chromatin fragment-IgG complexes in the mesangial matrix and glomerular basement membranes (GBM). The latter defines end-stage disease.Methodology/Principals
In the present study we determined the impact of antibodies to dsDNA, renal Dnase1 and matrix metalloprotease (MMP) mRNA levels and enzyme activities on early and late events in murine lupus nephritis. The major focus was to analyse if these factors were interrelated, and if changes in their expression explain basic processes accounting for lupus nephritis.Findings
Early phases of nephritis were associated with chromatin-IgG complex deposition in the mesangial matrix. A striking observation was that this event correlated with appearance of anti-dsDNA antibodies and mild or clinically silent nephritis. These events preceded down-regulation of renal Dnase1. Later, renal Dnase1 mRNA level and enzyme activity were reduced, while MMP2 mRNA level and enzyme activity increased. Reduced levels of renal Dnase1 were associated in time with deficient fragmentation of chromatin from dead cells. Large fragments were retained and accumulated in GBM. Also, since chromatin fragments are prone to stimulate Toll-like receptors in e.g. dendritic cells, this may in fact explain increased expression of MMPs.Significance
These scenarios may explain the basis for deposition of chromatin-IgG complexes in glomeruli in early and late stages of nephritis, loss of glomerular integrity and finally renal failure. 相似文献17.
18.
Ian Postuma Silva Bortolussi Nicoletta Protti Francesca Ballarini Piero Bruschi Laura Ciani Sandra Ristori Luigi Panza Cinzia Ferrari Laura Cansolino Saverio Altieri 《Reports of Practical Oncology and Radiotherapy》2016,21(2):123-128
Aim
Boron Neutron Capture Therapy (BNCT) is a binary hadrontherapy which exploits the neutron capture reaction in boron, together with a selective uptake of boronated substances by the neoplastic tissue. There is increasing evidence that future improvements in clinical BNCT will be triggered by the discovery of new boronated compounds, with higher selectivity for the tumor with respect to clinically used sodium borocaptate (BSH) and boronophenylalanine (BPA).Background
Therefore, a 10B quantification technique for biological samples is needed in order to evaluate the performance of new boronated formulations.Materials and methods
This article describes an improved neutron autoradiography set-up employing radiation sensitive films where the latent tracks are made visible by proper etching conditions.Results
Calibration curves for both liquid and tissue samples were obtained.Conclusions
The obtained calibration curves were adopted to set-up a mechanism to point out boron concentration in the whole sample. 相似文献19.
Sukanta Chowdhury Salah Uddin Khan Gary Crameri Jonathan H. Epstein Christopher C. Broder Ausraful Islam Alison J. Peel Jennifer Barr Peter Daszak Lin-Fa Wang Stephen P. Luby 《PLoS neglected tropical diseases》2014,8(11)