Publications by authors named "Jonas Blomberg"

88 Publications

ICTV Virus Taxonomy Profile: 2021.

J Gen Virol 2021 Dec;102(12)

Boston College, Chestnut Hill, MA 02467, USA.

Viruses in the family are found in a wide variety of vertebrate hosts. Enveloped virions are 80-100 nm in diameter with an inner core containing the viral genome and replicative enzymes. Core morphology is often characteristic for viruses within the same genus. Replication involves reverse transcription and integration into host cell DNA, resulting in a provirus. Integration into germline cells can result in a heritable provirus known as an endogenous retrovirus. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family , which is available at ictv.global/report/retroviridae.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/jgv.0.001712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8744268PMC
December 2021

Identification and characterization of ERV-W-like sequences in Platyrrhini species provides new insights into the evolutionary history of ERV-W in primates.

Mob DNA 2020 1;11. Epub 2020 Feb 1.

1Department of Life and Environmental Sciences, University of Cagliari, Cittadella Universitaria di Monserrato, SS554, Monserrato, Cagliari Italy.

Background: Endogenous Retroviruses (ERVs) constitute approximately 8% of every human genome and are relics of ancestral infections that affected the germ line cells. The ERV-W group contributed to primate physiology by providing an envelope protein (Syncytin-1) that has been adopted for placenta development in hominoids. Expression of Human ERV-W (HERV-W) sequences is investigated for a pathological role in various human diseases.

Results: We previously characterized ERV-W group genomic sequences in human and non-human Catarrhini species. We now investigated ERV-W-like sequences in the parvorder Platyrrhini, especially regarding two species with complete genome assemblies, namely marmoset () and squirrel monkey (). We identified in both species proviral sequences, annotated as ERV1-1 in respective genome assemblies, sharing high sequence similarities with Catarrhini ERV-W. A total of 130 relatively intact proviruses from the genomes of marmoset and squirrel monkey were characterized regarding their structural and evolutionarily relationships with Catarrhini ERV-W elements. Platyrrhini ERV-W sequences share several structural features with Catarrhini ERV-W elements and are closely related phylogenetically with the latter as well as with other ERV-W-related gammaretrovirus-like ERVs. The ERV-W group colonized Platyrrhini primates of both Callitrichidae and Atelidae lineages, with provirus formations having occurred mostly between 25 and 15 mya. Two LTR subgroups were associated with monophyletic proviral bodies. A region appears to be a sequence feature common to the ERV-W group: it harbors a putative intron sequence that is missing in some ERV-W loci, holding a putative ORF as well. The presence of a long portion was confirmed among all gammaretroviral ERV analyzed, suggesting a role in the latter biology. It is noteworthy that, contrary to Catarrhini ERV-W, there was no evidence of L1-mediated mobilization for Platyrrhini ERV-W sequences.

Conclusions: Our data establish that ERV-W is not exclusive to Catarrhini primates but colonized both parvorders of Simiiformes, providing further insight into the evolution of ERV-W and the colonization of primate genomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13100-020-0203-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6995185PMC
February 2020

Antibodies to Human Herpesviruses in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients.

Front Immunol 2019 14;10:1946. Epub 2019 Aug 14.

Gottfries Clinic AB, Mölndal, Sweden.

Myalgic encephalomyelitis, also referred to as chronic fatigue syndrome (ME/CFS) is a debilitating disease characterized by myalgia and a sometimes severe limitation of physical activity and cognition. It is exacerbated by physical and mental activity. Its cause is unknown, but frequently starts with an infection. The eliciting infection (commonly infectious mononucleosis or an upper respiratory infection) can be more or less well diagnosed. Among the human herpesviruses (HHV-1-8), HHV-4 (Epstein-Barr virus; EBV), HHV-6 (including HHV-6A and HHV-6B), and HHV-7, have been implicated in the pathogenesis of ME/CFS. It was therefore logical to search for serological evidence of past herpesvirus infection/reactivation in several cohorts of ME/CFS patients (all diagnosed using the Canada criteria). Control samples were from Swedish blood donors. We used whole purified virus, recombinant proteins, and synthetic peptides as antigens in a suspension multiplex immunoassay (SMIA) for immunoglobulin G (IgG). The study on herpesviral peptides based on antigenicity with human sera yielded novel epitope information. Overall, IgG anti-herpes-viral reactivities of ME/CFS patients and controls did not show significant differences. However, the high precision and internally controlled format allowed us to observe minor relative differences between antibody reactivities of some herpesviral antigens in ME/CFS versus controls. ME/CFS samples reacted somewhat differently from controls with whole virus HHV-1 antigens and recombinant EBV EBNA6 and EA antigens. We conclude that ME/CFS samples had similar levels of IgG reactivity as blood donor samples with HHV-1-7 antigens. The subtle serological differences should not be over-interpreted, but they may indicate that the immune system of some ME/CFS patients interact with the ubiquitous herpesviruses in a way different from that of healthy controls.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.01946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702656PMC
October 2020

Comprehensive Characterization of the Human Endogenous Retrovirus HERV-K(HML-6) Group: Overview of Structure, Phylogeny, and Contribution to the Human Genome.

J Virol 2019 08 30;93(16). Epub 2019 Jul 30.

Laboratory of Molecular Virology, Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy

Eight percent of the human genome is composed of human endogenous retroviruses (HERVs), remnants of ancestral germ line infections by exogenous retroviruses, which have been vertically transmitted as Mendelian characters. The HML-6 group, a member of the class II betaretrovirus-like viruses, includes several proviral loci with an increased transcriptional activity in cancer and at least two elements that are known for retaining an intact open reading frame and for encoding small proteins such as ERVK3-1, which is expressed in various healthy tissues, and HERV-K-MEL, a small Env peptide expressed in samples of cutaneous and ocular melanoma but not in normal tissues. We reported the distribution and genetic composition of 66 HML-6 elements. We analyzed the phylogeny of the HML-6 sequences and identified two main clusters. We provided the first description of a Rec domain within the sequence of 23 HML-6 elements. A Rec domain was also predicted within the ERVK3-1 transcript sequence, revealing its expression in various healthy tissues. Evidence about the context of insertion and colocalization of 19 HML-6 elements with functional human genes are also reported, including the sequence 16p11.2, whose 5' long terminal repeat overlapped the exon of one transcript variant of a cellular zinc finger upregulated and involved in hepatocellular carcinoma. The present work provides the first complete overview of the HML-6 elements in GRCh37(hg19), describing the structure, phylogeny, and genomic context of insertion of each locus. This information allows a better understanding of the genetics of one of the most expressed HERV groups in the human genome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.00110-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6675890PMC
August 2019

Investigation of somatic single nucleotide variations in human endogenous retrovirus elements and their potential association with cancer.

PLoS One 2019 1;14(4):e0213770. Epub 2019 Apr 1.

Department of Biochemistry & Molecular Medicine, George Washington University Medical Center, Washington, DC, United States of America.

Human endogenous retroviruses (HERVs) have been investigated for potential links with human cancer. However, the distribution of somatic nucleotide variations in HERV elements has not been explored in detail. This study aims to identify HERV elements with an over-representation of somatic mutations (hot spots) in cancer patients. Four HERV elements with mutation hotspots were identified that overlap with exons of four human protein coding genes. These hotspots were identified based on the significant over-representation (p<8.62e-4) of non-synonymous single-nucleotide variations (nsSNVs). These genes are TNN (HERV-9/LTR12), OR4K15 (HERV-IP10F/LTR10F), ZNF99 (HERV-W/HERV17/LTR17), and KIR2DL1 (MST/MaLR). In an effort to identify mutations that effect survival, all nsSNVs were further evaluated and it was found that kidney cancer patients with mutation C2270G in ZNF99 have a significantly lower survival rate (hazard ratio = 2.6) compared to those without it. Among HERV elements in the human non-protein coding regions, we found 788 HERVs with significantly elevated numbers of somatic single-nucleotide variations (SNVs) (p<1.60e-5). From this category the top three HERV elements with significantly over-represented SNVs are HERV-H/LTR7, HERV-9/LTR12 and HERV-L/MLT2. Majority of the SNVs in these 788 HERV elements are located in three DNA functional groups: long non-coding RNAs (lncRNAs) (60%), introns (22.2%) and transcriptional factor binding sites (TFBS) (14.8%). This study provides a list of mutational hotspots in HERVs, which could potentially be used as biomarkers and therapeutic targets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0213770PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6443178PMC
December 2019

Nomenclature for endogenous retrovirus (ERV) loci.

Retrovirology 2018 08 28;15(1):59. Epub 2018 Aug 28.

Biology Department, Boston College, Chestnut Hill, Massachusetts, 02467, USA.

Retroviral integration into germline DNA can result in the formation of a vertically inherited proviral sequence called an endogenous retrovirus (ERV). Over the course of their evolution, vertebrate genomes have accumulated many thousands of ERV loci. These sequences provide useful retrospective information about ancient retroviruses, and have also played an important role in shaping the evolution of vertebrate genomes. There is an immediate need for a unified system of nomenclature for ERV loci, not only to assist genome annotation, but also to facilitate research on ERVs and their impact on genome biology and evolution. In this review, we examine how ERV nomenclatures have developed, and consider the possibilities for the implementation of a systematic approach for naming ERV loci. We propose that such a nomenclature should not only provide unique identifiers for individual loci, but also denote orthologous relationships between ERVs in different species. In addition, we propose that-where possible-mnemonic links to previous, well-established names for ERV loci and groups should be retained. We show how this approach can be applied and integrated into existing taxonomic and nomenclature schemes for retroviruses, ERVs and transposable elements.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12977-018-0442-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6114882PMC
August 2018

Infection Elicited Autoimmunity and Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: An Explanatory Model.

Front Immunol 2018 15;9:229. Epub 2018 Feb 15.

Department of Clinical and Experimental Medicine, Division of Cell Biology, Linköping University, Linköping, Sweden.

Myalgic encephalomyelitis (ME) often also called chronic fatigue syndrome (ME/CFS) is a common, debilitating, disease of unknown origin. Although a subject of controversy and a considerable scientific literature, we think that a solid understanding of ME/CFS pathogenesis is emerging. In this study, we compiled recent findings and placed them in the context of the clinical picture and natural history of the disease. A pattern emerged, giving rise to an explanatory model. ME/CFS often starts after or during an infection. A logical explanation is that the infection initiates an autoreactive process, which affects several functions, including brain and energy metabolism. According to our model for ME/CFS pathogenesis, patients with a genetic predisposition and dysbiosis experience a gradual development of B cell clones prone to autoreactivity. Under normal circumstances these B cell offsprings would have led to tolerance. Subsequent exogenous microbial exposition (triggering) can lead to comorbidities such as fibromyalgia, thyroid disorder, and orthostatic hypotension. A decisive infectious trigger may then lead to immunization against autoantigens involved in aerobic energy production and/or hormone receptors and ion channel proteins, producing postexertional malaise and ME/CFS, affecting both muscle and brain. In principle, cloning and sequencing of immunoglobulin variable domains could reveal the evolution of pathogenic clones. Although evidence consistent with the model accumulated in recent years, there are several missing links in it. Hopefully, the hypothesis generates testable propositions that can augment the understanding of the pathogenesis of ME/CFS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2018.00229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5818468PMC
March 2019

Distinction between serological responses following tick-borne encephalitis virus (TBEV) infection vs vaccination, Sweden 2017.

Euro Surveill 2018 01;23(3)

Laboratory of Clinical Microbiology, Uppsala University Hospital, Uppsala, Sweden.

Tick-borne encephalitis virus (TBEV) is an important European vaccine-preventable pathogen. Discrimination of vaccine-induced antibodies from those elicited by infection is important. We studied anti-TBEV IgM/IgG responses, including avidity and neutralisation, by multiplex serology in 50 TBEV patients and 50 TBEV vaccinees. Infection induced antibodies reactive to both whole virus (WV) and non-structural protein 1 (NS1) in 48 clinical cases, whereas 47 TBEV vaccinees had WV, but not NS1 antibodies, enabling efficient discrimination of infection/vaccination.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2807/1560-7917.ES.2018.23.3.17-00838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792698PMC
January 2018

HERV-W group evolutionary history in non-human primates: characterization of ERV-W orthologs in Catarrhini and related ERV groups in Platyrrhini.

BMC Evol Biol 2018 01 19;18(1). Epub 2018 Jan 19.

Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy.

Background: The genomes of all vertebrates harbor remnants of ancient retroviral infections, having affected the germ line cells during the last 100 million years. These sequences, named Endogenous Retroviruses (ERVs), have been transmitted to the offspring in a Mendelian way, being relatively stable components of the host genome even long after their exogenous counterparts went extinct. Among human ERVs (HERVs), the HERV-W group is of particular interest for our physiology and pathology. A HERV-W provirus in locus 7q21.2 has been coopted during evolution to exert an essential role in placenta, and the group expression has been tentatively linked to Multiple Sclerosis and other diseases. Following up on a detailed analysis of 213 HERV-W insertions in the human genome, we now investigated the ERV-W group genomic spread within primate lineages.

Results: We analyzed HERV-W orthologous loci in the genome sequences of 12 non-human primate species belonging to Simiiformes (parvorders Catarrhini and Platyrrhini), Tarsiiformes and to the most primitive Prosimians. Analysis of HERV-W orthologous loci in non-human Catarrhini primates revealed species-specific insertions in the genomes of Chimpanzee (3), Gorilla (4), Orangutan (6), Gibbon (2) and especially Rhesus Macaque (66). Such sequences were acquired in a retroviral fashion and, in the majority of cases, by L1-mediated formation of processed pseudogenes. There were also a number of LTR-LTR homologous recombination events that occurred subsequent to separation of Catarrhini sub-lineages. Moreover, we retrieved 130 sequences in Marmoset and Squirrel Monkeys (family Cebidae, Platyrrhini parvorder), identified as ERV1-1_CJa based on RepBase annotations, which appear closely related to the ERV-W group. Such sequences were also identified in Atelidae and Pitheciidae, representative of the other Platyrrhini families. In contrast, no ERV-W-related sequences were found in genome sequence assemblies of Tarsiiformes and Prosimians.

Conclusions: Overall, our analysis now provides a detailed picture of the ERV-W sequences colonization of the primate lineages genomes, revealing the exact dynamics of ERV-W locus formations as well as novel insights into the evolution and origin of the group.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12862-018-1125-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5775608PMC
January 2018

Identification of a novel HERV-K(HML10): comprehensive characterization and comparative analysis in non-human primates provide insights about HML10 proviruses structure and diffusion.

Mob DNA 2017 2;8:15. Epub 2017 Nov 2.

Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy.

Background: About half of the human genome is constituted of transposable elements, including human endogenous retroviruses (HERV). HERV sequences represent the 8% of our genetic material, deriving from exogenous infections occurred millions of years ago in the germ line cells and being inherited by the offspring in a Mendelian fashion. HERV-K elements (classified as HML1-10) are among the most studied HERV groups, especially due to their possible correlation with human diseases. In particular, the HML10 group was reported to be upregulated in persistent HIV-1 infected cells as well as in tumor cells and samples, and proposed to have a role in the control of host genes expression. An individual HERV-K(HML10) member within the major histocompatibility complex C4 gene has even been studied for its possible contribution to type 1 diabetes susceptibility. Following a first characterization of the HML10 group at the genomic level, performed with the innovative software RetroTector, we have characterized in detail the 8 previously identified HML10 sequences present in the human genome, and an additional HML10 partial provirus in chromosome 1p22.2 that is reported here for the first time.

Results: Using a combined approach based on RetroTector software and a traditional Genome Browser Blat search, we identified a novel HERV-K(HML10) sequence in addition to the eight previously reported in the human genome GRCh37/hg19 assembly. We fully characterized the nine HML10 sequences at the genomic level, including their classification in two types based on both structural and phylogenetic characteristics, a detailed analysis of each HML10 nucleotide sequence, the first description of the presence of an Env Rec domain in the type II HML10, the estimated time of integration of individual members and the comparative map of the HML10 proviruses in non-human primates.

Conclusions: We performed an unambiguous and exhaustive analysis of the nine HML10 sequences present in GRCh37/hg19 assembly, useful to increase the knowledge of the group's contribution to the human genome and laying the foundation for a better understanding of the potential physiological effects and the tentative correlation of these sequences with human pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13100-017-0099-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5667498PMC
November 2017

Compensating for cross-reactions using avidity and computation in a suspension multiplex immunoassay for serotyping of Zika versus other flavivirus infections.

Med Microbiol Immunol 2017 Oct 29;206(5):383-401. Epub 2017 Aug 29.

Section of Clinical Microbiology, Department of Medical Sciences, Uppsala Academic Hospital, Uppsala University, 751 85, Uppsala, Sweden.

The recent spread of Zika virus (ZIKV) in the Americas and Asia necessitates an increased preparedness for improved maternal and perinatal health and blood safety. However, serological cross-reactions, especially to Dengue virus (DENV), complicate ZIKV antibody serodiagnosis. A novel "pan-Flavi" suspension multiplex immunoassay (PFSMIA) using 25 antigens, whole virus (WV), non-structural protein 1 (NS1), and envelope (E) proteins, from 7 zoonotic flaviviruses for specific detection of ZIKV and DENV IgM and IgG was developed. Patterns of antibody cross-reactivity, avidity, and kinetics were established in 104 sera from returning travelers with known ZIKV and DENV infections. PFSMIA gave IgM- and IgG-sensitivities for both viruses of 96-100%, compared to an immunofluorescence assay. Main IgM cross-reactions were to NS1, for IgG to the E and WV antigens. Infecting virus yielded reactivity to several antigens of the homologous virus, while cross-reactions tended to occur only to a single antigen from heterologous virus(es). A specificity-enhancing computer procedure took into account antibody isotype, number of antibody-reactive antigens per virus, avidity, average degree of cross-reactivity to heterologous flavivirus antigens, and reactivity changes in serial sera. It classified all 50 cases correctly. Applied to sera from 200 pregnant women and 173 blood donors from Sweden, one blood donor was found ZIKV NS1 IgM positive, and another as ZIKV NS1 IgG positive. These samples did not react with other ZIKV antigens and were thereby judged as false-positives. PFSMIA provided sensitive and specific ZIKV and DENV serology, warranting high-throughput serological surveillance and a minimized need for laborious and expensive virus neutralization assays.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00430-017-0517-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5599479PMC
October 2017

Serogrouping and seroepidemiology of North European hantaviruses using a novel broadly targeted synthetic nucleoprotein antigen array.

Infect Ecol Epidemiol 2017 26;7(1):1350086. Epub 2017 Jul 26.

Section of Clinical Microbiology, Department of Medical Sciences, Uppsala University, Uppsala, Sweden.

: Hantaviruses are globally distributed zoonotic pathogens. Great diversity and high antigenic cross-reactivity makes diagnosis by traditional methods cumbersome. : 'Megapeptides', 119-120-mers from the amino terminus of the nucleoprotein of 16 hantaviruses, representing the four major branches of the hantavirus phylogenetic tree, were utilized in a novel IgG-based hantavirus suspension multiplex immunoassay (HSMIA) for detection of past hantavirus infections in 155 North European human samples. We compared HSMIA with established EIAs and focus reduction neutralization test (FRNT). : The Puumala hantavirus (PUUV) component in the HSMIA gave concordant results with a PUUV IgG EIA in 142 sera from Northern Sweden (of which 31 were EIA positive, 7 borderline and 104 EIA negative, sensitivity 30/31 = 97%, specificity 104/ 104 = 100%, 134/135 = 99% concordance), with another immunoassay in 40 PUUV IgG positive sera from Finland (36/40 = 90% sensitivity), and was concordant in 8 of 11 cases with PUUV and DOBV neutralization titers, respectively. Two major IgG reactivity patterns were found: (i) a PUUV-specific pattern covering phylogroup IV and its serogroups B and C; and (ii) a Dobrava virus (DOBV)-specific pattern, covering the serogroup A portion of phylogroup III. In addition, we found several minor patterns with reactivity to only one or two megapeptides indicating additional hantaviruses infecting humans in the Swedish and Finnish populations. : The broadly reactive and rational HSMIA yielded results highly correlated with the established PUUV EIAs and the NT results. It is a sensitive and specific assay, which will be suited for efficient serosurveillance of hantaviruses in humans. Its use in animals should be further investigated.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/20008686.2017.1350086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5549826PMC
July 2017

Decreased HHV-6 IgG in Alzheimer's Disease.

Front Neurol 2017 20;8:40. Epub 2017 Feb 20.

Department of Medical Sciences, Uppsala University , Uppsala , Sweden.

Human herpesviruses have previously been implicated in the pathogenesis of Alzheimer's disease (AD) but whether they are causal, facilitating, or confounding factors is yet to be established. A total of 50 AD subjects and 52 non-demented (ND) controls were analyzed in a multiplex assay for IgG reactivity toward herpes simplex virus (HSV), varicella zoster virus (VZV), cytomegalovirus (CMV), and human herpesvirus 6 (HHV-6). The HHV-6 IgG reactivity was significantly lower in AD subjects compared to ND controls, whereas there were no differences in HSV, VZV, or CMV antibody levels between the groups. Analysis of peripheral blood mononuclear cells with a subtype-specific HHV-6 PCR revealed no signs of reactivation, as AD and ND subjects presented with comparable HHV-6 DNA levels in PBMCs, and all positive samples were of subtype B. Whether HHV-6 is a factor in AD remains to be elucidated in future studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fneur.2017.00040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5316842PMC
February 2017

Duplex detection of the Mycobacterium tuberculosis complex and medically important non-tuberculosis mycobacteria by real-time PCR based on the rnpB gene.

APMIS 2016 Nov 28;124(11):991-995. Epub 2016 Sep 28.

Section of Clinical Bacteriology, Department of Medical Sciences, Uppsala University, Uppsala, Sweden.

A duplex real-time PCR based on the rnpB gene was developed for Mycobacterium spp. The assay was specific for the Mycobacterium tuberculosis complex (MTB) and also detected all 19 tested species of non-tuberculous mycobacteria (NTM). The assay was evaluated on 404 clinical samples: 290 respiratory samples and 114 from tissue and other non-respiratory body sites. M. tuberculosis was detected by culture in 40 samples and in 30 samples by the assay. The MTB assay showed a sensitivity similar to Roche Cobas Amplicor MTB-PCR (Roche Molecular Systems, Pleasanton, CA, USA). There were only nine samples with non-tuberculous mycobacteria detected by culture. Six of them were detected by the PCR assay.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/apm.12598DOI Listing
November 2016

Contribution of type W human endogenous retroviruses to the human genome: characterization of HERV-W proviral insertions and processed pseudogenes.

Retrovirology 2016 09 9;13(1):67. Epub 2016 Sep 9.

Department of Life and Environmental Sciences, University of Cagliari, Cittadella Universitaria di Monserrato SS554, 09042, Monserrato, Cagliari, Italy.

Background: Human endogenous retroviruses (HERVs) are ancient sequences integrated in the germ line cells and vertically transmitted through the offspring constituting about 8 % of our genome. In time, HERVs accumulated mutations that compromised their coding capacity. A prominent exception is HERV-W locus 7q21.2, producing a functional Env protein (Syncytin-1) coopted for placental syncytiotrophoblast formation. While expression of HERV-W sequences has been investigated for their correlation to disease, an exhaustive description of the group composition and characteristics is still not available and current HERV-W group information derive from studies published a few years ago that, of course, used the rough assemblies of the human genome available at that time. This hampers the comparison and correlation with current human genome assemblies.

Results: In the present work we identified and described in detail the distribution and genetic composition of 213 HERV-W elements. The bioinformatics analysis led to the characterization of several previously unreported features and provided a phylogenetic classification of two main subgroups with different age and structural characteristics. New facts on HERV-W genomic context of insertion and co-localization with sequences putatively involved in disease development are also reported.

Conclusions: The present work is a detailed overview of the HERV-W contribution to the human genome and provides a robust genetic background useful to clarify HERV-W role in pathologies with poorly understood etiology, representing, to our knowledge, the most complete and exhaustive HERV-W dataset up to date.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12977-016-0301-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016936PMC
September 2016

Serology in the Digital Age: Using Long Synthetic Peptides Created from Nucleic Acid Sequences as Antigens in Microarrays.

Microarrays (Basel) 2016 Aug 10;5(3). Epub 2016 Aug 10.

Section of Clinical Virology, Department of Medical Sciences, Uppsala University, Uppsala 751 85, Sweden.

Background: Antibodies to microbes, or to autoantigens, are important markers of disease. Antibody detection (serology) can reveal both past and recent infections. There is a great need for development of rational ways of detecting and quantifying antibodies, both for humans and animals. Traditionally, serology using synthetic antigens covers linear epitopes using up to 30 amino acid peptides.

Methods: We here report that peptides of 100 amino acids or longer ("megapeptides"), designed and synthesized for optimal serological performance, can successfully be used as detection antigens in a suspension multiplex immunoassay (SMIA). Megapeptides can quickly be created just from pathogen sequences. A combination of rational sequencing and bioinformatic routines for definition of diagnostically-relevant antigens can, thus, rapidly yield efficient serological diagnostic tools for an emerging infectious pathogen.

Results: We designed megapeptides using bioinformatics and viral genome sequences. These long peptides were tested as antigens for the presence of antibodies in human serum to the filo-, herpes-, and polyoma virus families in a multiplex microarray system. All of these virus families contain recently discovered or emerging infectious viruses.

Conclusion: Long synthetic peptides can be useful as serological diagnostic antigens, serving as biomarkers, in suspension microarrays.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5040969PMC
http://dx.doi.org/10.3390/microarrays5030022DOI Listing
August 2016

Dynamic and selective HERV RNA expression in neuroblastoma cells subjected to variation in oxygen tension and demethylation.

APMIS 2016 Jan-Feb;124(1-2):140-9

Section of Virology, Department of Medical Sciences, Uppsala University, Uppsala, Sweden.

We studied HERV expression in cell lines after hypoxia, mitogenic stimulation, and demethylation, to better understand if hypoxia may play a role in ERV activation also within the nervous system, as represented by neuroblastoma cell lines. The level of RNA of four human ERV groups (HERVs) (HERVE, I/T, H, and W), and three housekeeping genes, of different cell lines including A549, COS-1, Namalwa, RD-L and Vero-E6, as well as human neuroblastoma cell lines SH-SY5Y, SK-N-DZ, and SK-N-AS were studied using reverse transcription and real-time quantitative PCR (QPCR). During the course of recovery from hypoxia a pronounced and selective activation of RNA expression of HERVW-like sequences, but not of HERVE, I/T, H, and three housekeeping genes, was found in the neuroblastoma cell lines, most pronounced in SK-N-DZ. In the SK-N-DZ cell line, we also tested the expression of HERVs after chemical treatments. HERVW-like sequences were selectively upregulated by 5-azacytidine, a demethylating agent. Some HERVW loci seem especially responsive to hypoxia and demethylation. HERV expression in neuroblastoma cells is selectively and profoundly influenced by some physiological and chemical stimuli.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/apm.12494DOI Listing
June 2016

Classification and characterization of human endogenous retroviruses; mosaic forms are common.

Retrovirology 2016 Jan 22;13. Epub 2016 Jan 22.

Department of Medical Sciences, Uppsala University Hospital, Dag Hammarskjölds Väg 17, Uppsala, 751 85, Sweden.

Background: Human endogenous retroviruses (HERVs) represent the inheritance of ancient germ-line cell infections by exogenous retroviruses and the subsequent transmission of the integrated proviruses to the descendants. ERVs have the same internal structure as exogenous retroviruses. While no replication-competent HERVs have been recognized, some retain up to three of four intact ORFs. HERVs have been classified before, with varying scope and depth, notably in the RepBase/RepeatMasker system. However, existing classifications are bewildering. There is a need for a systematic, unifying and simple classification. We strived for a classification which is traceable to previous classifications and which encompasses HERV variation within a limited number of clades.

Results: The human genome assembly GRCh 37/hg19 was analyzed with RetroTector, which primarily detects relatively complete Class I and II proviruses. A total of 3173 HERV sequences were identified. The structure of and relations between these proviruses was resolved through a multi-step classification procedure that involved a novel type of similarity image analysis ("Simage") which allowed discrimination of heterogeneous (noncanonical) from homogeneous (canonical) HERVs. Of the 3173 HERVs, 1214 were canonical and segregated into 39 canonical clades (groups), belonging to class I (Gamma- and Epsilon-like), II (Beta-like) and III (Spuma-like). The groups were chosen based on (1) sequence (nucleotide and Pol amino acid), similarity, (2) degree of fit to previously published clades, often from RepBase, and (3) taxonomic markers. The groups fell into 11 supergroups. The 1959 noncanonical HERVs contained 31 additional, less well-defined groups. Simage analysis revealed several types of mosaicism, notably recombination and secondary integration. By comparing flanking sequences, LTRs and completeness of gene structure, we deduced that some noncanonical HERVs proliferated after the recombination event. Groups were further divided into envelope subgroups (altogether 94) based on sequence similarity and characteristic "immunosuppressive domain" motifs. Intra and inter(super)group, as well as intraclass, recombination involving envelope genes ("env snatching") was a common event. LTR divergence indicated that HERV-K(HML2) and HERVFC had the most recent integrations, HERVL and HUERSP3 the oldest.

Conclusions: A comprehensive HERV classification and characterization approach was undertaken. It should be applicable for classification of all ERVs. Recombination was common among HERV ancestors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12977-015-0232-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4724089PMC
January 2016

Development of single-tube nested real-time PCR assays with long internally quenched probes for detection of norovirus genogroup II.

Biotechniques 2016 Jan 1;60(1):28-34. Epub 2016 Jan 1.

Section of Clinical Virology, Department of Medical Sciences, Uppsala University, Sweden.

The high sequence variation of RNA viruses necessitates use of degenerate primers and probes or multiple primers and probes in molecular diagnostic assays. We showed previously that PCR amplification in two rounds, first with long target-specific primers and then with short generic primers, followed by detection using long probes, can tolerate sequence variation. Here we demonstrate that long primers and probes of up to 56 nucleotides can also be applied in real-time PCR for the detection of norovirus genogroup II with improved sensitivity. Probe design (method of incorporating quenchers, use of Zen internal quencher or traditional quenchers) greatly affects the sensitivity of the real-time PCR assays.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2144/000114370DOI Listing
January 2016

Microsphere-based antibody assays for human parvovirus B19V, CMV and T. gondii.

BMC Infect Dis 2016 Jan 8;16. Epub 2016 Jan 8.

Virology, University of Helsinki, Haartmaninkatu 3, FI-00290, Helsinki, Finland.

Background: Human parvovirus B19 (B19V), cytomegalovirus (CMV) and Toxoplasma gondii (T. gondii) may cause intrauterine infections with potentially severe consequences to the fetus. Current serodiagnosis of these infections is based on detection of antibodies most often by EIA and individually for each pathogen. We developed singleplex and multiplex microsphere-based Suspension Immuno Assays (SIAs) for the simultaneous detection of IgG antibodies against B19V, CMV and T. gondii.

Methods: We tested the performances of SIAs as compared to in-house and commercial reference assays using serum samples from well-characterized cohorts.

Results: The IgG SIAs for CMV and T. gondii showed good concordance with the corresponding Vidas serodiagnostics. The B19V IgG SIA detected IgG in all samples collected >10 days after onset of symptoms and showed high concordance with EIAs (in-house and Biotrin). The serodiagnostics for these three pathogens performed well in multiplex format.

Conclusions: We developed singleplex and multiplex IgG SIAs for the detection of anti-B19V, -CMV and -T. gondii antibodies. The SIAs were highly sensitive and specific, and had a wide dynamic range. These components thus should be suitable for construction of a multiplex test for antibody screening during pregnancy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12879-015-1194-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4706663PMC
January 2016

Detection of rotavirus using padlock probes and rolling circle amplification.

PLoS One 2014 4;9(11):e111874. Epub 2014 Nov 4.

Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Solna, Sweden.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0111874PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4219791PMC
December 2015

Transmission of hepatitis C virus among intravenous drug users in the Uppsala region of Sweden.

Infect Ecol Epidemiol 2014 Jan 15;4. Epub 2014 Jan 15.

Section of Clinical Virology, Department of Medical Sciences, Uppsala University, Uppsala, Sweden.

Background: 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.

Method: Eighty-two serum samples were collected from IDUs in Uppsala County. Our reverse transcription nested polymerase chain reaction (RT-nested PCR) and sequencing method enabled a comprehensive genetic analysis for a broad spectrum of genotypes of two relatively conserved regions, NS5B and NS3, that encodes for the viral polymerase and protease, respectively. HCV RNA in serum samples was amplified and sequenced with in-house primers. Sequence similarities between individuals and subgroups were analyzed with maximum likelihood (ML) phylogenetic trees. Published HCV reference sequences from other geographic regions and countries were also included for clarity.

Results: Phylogenetic analysis was possible for 59 NS5B (72%) and 29 NS3 (35%) sequences from Uppsala patients. Additionally, we also included 15 NS3 sequences from Örebro patients, making a total of 44 NS3 sequences for the analysis. By analyzing the NS3 sequences, two transmission sets were found between the IDUs (>98% sequence identity), with one set consisting of two individuals and another set consisting of three individuals. In addition, the phylogenetic analysis done with our serum samples displayed clusters that distinguished them from the reference sequences.

Conclusion: Our method seems to enable us to trace the HCV transmission between IDUs. Furthermore, the method is fairly independent of the time of infection because the method uses relatively conserved HCV sequence regions (i.e. NS5B and NS3).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3402/iee.v4.22251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3895264PMC
January 2014

Epitopes of microbial and human heat shock protein 60 and their recognition in myalgic encephalomyelitis.

PLoS One 2013 28;8(11):e81155. Epub 2013 Nov 28.

Section of Clinical Microbiology, Department of Medical Sciences, Uppsala University, Uppsala, Sweden.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0081155PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3842916PMC
December 2014

Quantitative fucK gene polymerase chain reaction on sputum and nasopharyngeal secretions to detect Haemophilus influenzae pneumonia.

Diagn Microbiol Infect Dis 2013 Jun 27;76(2):141-6. Epub 2013 Mar 27.

Section of Clinical Bacteriology, Department of Medical Sciences, Uppsala University, S-75185, Uppsala, Sweden.

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 patient's 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.diagmicrobio.2013.02.015DOI Listing
June 2013

Conserved structure and inferred evolutionary history of long terminal repeats (LTRs).

Mob DNA 2013 Feb 1;4(1). Epub 2013 Feb 1.

Section of Virology, Department of Medical Sciences, Uppsala University, Uppsala, Sweden.

Background: 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.

Results: Hidden Markov models (HMM) were created for 11 clades of LTRs belonging to Retroviridae (class III retroviruses), animal Metaviridae (Gypsy/Ty3) elements and plant Pseudoviridae (Copia/Ty1) elements, complementing our work with Orthoretrovirus HMMs. The great variation in LTR length of plant Metaviridae and the few divergent animal Pseudoviridae prevented building HMMs from both of these groups.Animal Metaviridae LTRs had the same conserved motifs as retroviral LTRs, confirming that the two groups are closely related. The conserved motifs were the short inverted repeats (SIRs), integrase recognition signals (5´TGTTRNR…YNYAACA 3´); the polyadenylation signal or AATAAA motif; a GT-rich stretch downstream of the polyadenylation signal; and a less conserved AT-rich stretch corresponding to the core promoter element, the TATA box. Plant Pseudoviridae LTRs differed slightly in having a conserved TATA-box, TATATA, but no conserved polyadenylation signal, plus a much shorter R region.The sensitivity of the HMMs for detection in genomic sequences was around 50% for most models, at a relatively high specificity, suitable for genome screening.The HMMs yielded consensus sequences, which were aligned by creating an HMM model (a 'Superviterbi' alignment). This yielded a phylogenetic tree that was compared with a Pol-based tree. Both LTR and Pol trees supported monophyly of retroviruses. In both, Pseudoviridae was ancestral to all other LTR retrotransposons. However, the LTR trees showed the chromovirus portion of Metaviridae clustering together with Pseudoviridae, dividing Metaviridae into two portions with distinct phylogeny.

Conclusion: The HMMs clearly demonstrated a unitary conserved structure of LTRs, supporting that they arose once during evolution. We attempted to follow the evolution of LTRs by tracing their functional foundations, that is, acquisition of RNAse H, a combined promoter/ polyadenylation site, integrase, hairpin priming and the primer binding site (PBS). Available information did not support a simple evolutionary chain of events.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1759-8753-4-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3601003PMC
February 2013

Quantitative and multiplex detection of pathogenic fungi using padlock probes, generic qPCR, and suspension array readout.

Methods Mol Biol 2013 ;968:105-18

School of Health and Social Studies, Dalarna University, Falun, Sweden.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-62703-257-5_8DOI Listing
June 2013

Analyses of pig genomes provide insight into porcine demography and evolution.

Nature 2012 Nov;491(7424):393-8

Animal Breeding and Genomics Centre, Wageningen University, De Elst 1, 6708 WD, Wageningen, The Netherlands.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature11622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3566564PMC
November 2012

Unexpected diversity and expression of avian endogenous retroviruses.

mBio 2012 Oct 16;3(5):e00344-12. Epub 2012 Oct 16.

Department of Biology, Johns Hopkins University, Baltimore, Maryland, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mBio.00344-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3482500PMC
October 2012

Variation-tolerant capture and multiplex detection of nucleic acids: application to detection of microbes.

J Clin Microbiol 2012 Oct 18;50(10):3208-15. Epub 2012 Jul 18.

Section of Clinical Virology, Department of Medical Sciences, Uppsala University Hospital, Uppsala, Sweden.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JCM.06382-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3457464PMC
October 2012
-->