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Development of real-time PCRs for detection and quantitation of human MMTV-Iike (HML) sequences HML expression in human tissues
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. (Blomberg)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. (Blomberg)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. (Blomberg)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. (Blomberg)
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2006 (English)In: Journal of Virological Methods, ISSN 0166-0934, E-ISSN 1879-0984, Vol. 136, no 1-2, 83-92 p.Article in journal (Refereed) Published
Abstract [en]

The human genome contains around 1000 betaretrovirus-like copies, human mouse mammary tumour virus (MMTV)-like (HML) groups 1 - 10, also referred to as human endogenous retroviruts "HERV-K". Despite many efforts, it is not established whether betaretroviruses, exo- or endogenous, are involved in the etiology of breast cancer, or other cancer diseases, in humans. Quantitative real-time PCR (QPCR) TaqMan((R))-based assays for HML groups 1-7, targeting the conserved reverse transcriptase (RT) and integrase (IN) domains of the pol gene were designed. Plasmids containing the entire pol gene of HML 1-7 were used as standards. The RT and IN based QPCRs could detect 10(0)-10(3) copies per PCR reaction of the plasmids. However, not all plasmids gave a signal in both RT and IN QPCRs, probably due to mismatches. Furthermore, RT and IN based HML6 specific QPCRs were developed. They were specific for amplification of transcripts for the whole HML6 group. The methods allow the monitoring in body fluids and tissues of expression of a wide range of betaretrovirus-like sequences. Betaretrovirus-like RNA was studied in normal human tissues and of HML6 in brains of multiple sclerosis (MS) patients. Brain, adrenal gland and testis had a high betaretrovirus-like expression. Multiple sclerosis plaques contained the same HML6 RNA concentration as control tissue. These assays are expected to enhance studies on involvement of betaretroviruses in physiology and disease.

Place, publisher, year, edition, pages
2006. Vol. 136, no 1-2, 83-92 p.
Keyword [en]
HML, endogenous retrovirus, betaretrovirus, real-time PCR, expression, transcription
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:uu:diva-82749DOI: 10.1016/j.jviromet.2006.04.005ISI: 000239735200013PubMedID: 16713632OAI: oai:DiVA.org:uu-82749DiVA: diva2:110655
Available from: 2008-06-25 Created: 2008-06-25 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Endogenous Retroviral RNA Expression in Humans
Open this publication in new window or tab >>Endogenous Retroviral RNA Expression in Humans
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Human endogenous retroviruses (HERVs) constitute about 8% of the human genome. There are around 4000 pol-containing retroviral integrations in the human genome, which makes it impractical to measure each of them separately. Therefore we developed a set of degenerate real time PCRs to detect major groups bearing sequence similarities to gammaretroviruses, one of the largest groups of human endogenous retrovirus, and betaretroviruses, some of which have integrated into the human genome most recently and which remain the most intact.

It was found that, although both gammaretroviral and betaretroviral RNAs were broadly expressed in various healthy tissues including reproductive tissues and brain, a differential expression pattern was observed. My work further revealed that HERVE and HERVW, two gammaretroviral sequences, were ubiquitously and highly expressed in pathologic and normal female reproductive tissues with tissue specific patterns. Expression of HERVE was higher in endometriotic tissue than in normal endometrium. HERVE and HERVW RNAs were higher in normal ovarian tissue than in ovarian cancer. Besides these tissue- and neoplasia-related differences, there were wide differences in HERV expression among individuals. Next, a selective pattern of HERVW upregulation was demonstrated in SK-N-DZ, a neuroblastoma cell line, upon re-oxygenation after a period of hypoxia or with 5-azacytidine, a demethylating agent. Furthermore, broad and high expressions of gammaretrovirus-like transcripts in different brain areas analyzed were identified. The expression levels were variable among different donors.

In conclusion a ubiquitous HERV expression was observed in tissues and cell lines, with various patterns. At this stage the data are not sufficient to conclude whether HERV has any physiological or pathological roles in humans. However, their differential expression patterns are compatible with functional roles of HERV in humans.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 59 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 274
Keyword
Microbiology, Human endogenous retrovirus (HERV), HERVE, HERVW, betaretrovirus, gammaretrovirus, RNA expression, real time PCR, endometrium, endometriosis, brain tissue, reproductive tissue, ovarian cancer, ovary, neuroblastoma cell line, Mikrobiologi
Identifiers
urn:nbn:se:uu:diva-8213 (URN)978-91-554-6967-2 (ISBN)
Public defence
2007-10-12, Hörsalen, Baktlab ing D1, Dag Hammarskjölds Vag 17, 75185 Akademiska Sjukhuset, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2007-09-21 Created: 2007-09-21 Last updated: 2009-05-08Bibliographically approved
2. Detection and Quantification of Variable Viral RNA by Real-Time PCR Assays
Open this publication in new window or tab >>Detection and Quantification of Variable Viral RNA by Real-Time PCR Assays
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

As the area of nucleic acid based technologies develops, so will our understanding of how structural variations in DNA and RNA pathogens are associated with disease. The overall goal of this thesis is the development of broadly targeted measurement techniques for variable viral RNA by Real-Time PCR (here referred to as quantitative reverse transcriptase PCR, QRT-PCR).

In papers I & II, broadly targeted and specific QRT-PCRs were used to study expression of endogenous and exogenous betaretrovirus sequences in human tissues. Results from human tissues demonstrated endogenous betaretrovirus expression in a tissue-specific manner, highest in reproductive tissues. Despite the high sensitivity, no exogenous betaretrovirus was found in human breast cancer samples. The limits of primer and probe degeneracy for detection of a diverse set of retroviral sequences was evaluated. These methods are useful for further investigations on the pathophysiological contribution(s) of endogenous betaretrovirus and to investigate whether an exogenous betaretrovirus is involved in human breast cancer.

In papers III & IV, we developed and applied broadly targeted one-step QRT-PCRs for influenza viruses and coronaviruses. In addition to the generic primers, two novel probe design strategies were used in order to be able to broadly amplify these diverse sets of viruses: A triplex system for simultaneous detection and quantification of influenza A, B and C (3QRT-PCR and further developed 3QRT-PCR-MegB; where MegB stands for MegaBeacon) based on TaqMan® and MegB probes, and a pan-CoV QRT-PCR, based on three TaqMan® probes i.e., degeneracy was distributed on three probes. Probe fault tolerance was thus increased in two ways, either with short probes with/without locked nucleic acid (LNA) nucleotides concentrated to conserved stretches, or with long probes (MegB), compensating mismatching positions with many matching ones. Clinical samples, negative by antigen detection with immunofluorescence (IFA), were influenza A positive with 3QPCR-MegB. Avian pooled samples, negative with an earlier pan-CoV QPCR, came out positive with the triple-probe system. Assay evaluation with clinical samples and reference strains revealed good clinical diagnostic potential.

Thus, the thesis describes several strategies to counteract sequence variation of RNA viruses and describes a set of broadly targeted QRT-PCRs useful for scientific screening or diagnostics of betaretroviruses and respiratory viruses.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 67 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 365
Keyword
Virology, Diagnosis of Viral RNA, QPCR, RNA virus, broadly targeted PCR, probe design strategy, Infectious diseases
Research subject
Medicine
Identifiers
urn:nbn:se:uu:diva-9193 (URN)978-91-554-7251-1 (ISBN)
Public defence
2008-09-05, Hörsalen, Baktlab ing D1, Dag Hammarskjölds väg 17, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2008-08-15 Created: 2008-08-15 Last updated: 2011-02-28Bibliographically approved

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Muradrasoli, ShamanHu, LijuanBlikstad, VidarBlomberg, Jonas

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