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  • 51.
    Mohamed, Nahla
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Sciences.
    Belák, Sándor
    Hedlund, Kjell-Olof
    Blomberg, Jonas
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Sciences.
    Experience from the development of a diagnostic single tube real-time PCR for human caliciviruses, Norovirus genogroups I and II.2006In: J Virol Methods, ISSN 0166-0934, Vol. 132, no 1-2, p. 69-76Article in journal (Refereed)
  • 52.
    Mohamed, Nahla
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Sciences.
    Elfaitouri, Amal
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Sciences.
    Fohlman, Jan
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Sciences.
    Friman, Göran
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Sciences.
    Blomberg, Jonas
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Sciences.
    A sensitive and quantitative single-tube real-time reverse transcriptase-PCR for detection of enteroviral RNA.2004In: J Clin Virol, ISSN 1386-6532, Vol. 30, no 2, p. 150-6Article in journal (Refereed)
  • 53.
    Muradrasoli, Shaman
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Forsman, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Hu, Lijuan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Blikstad, Vidar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Development of real-time PCRs for detection and quantitation of human MMTV-Iike (HML) sequences HML expression in human tissues2006In: Journal of Virological Methods, ISSN 0166-0934, E-ISSN 1879-0984, Vol. 136, no 1-2, p. 83-92Article in journal (Refereed)
    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.

  • 54. Nilsson, A-L
    et al.
    Vaziri-Sani, F.
    Broberg, P.
    Elfaitouri, A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Pipkorn, R.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Ivarsson, S-A
    Larsson, H. Elding
    Lernmark, A.
    Serological Evaluation of Possible Exposure to Ljungan Virus and Related Parechovirus in Autoimmune (Type 1) Diabetes in Children2015In: Journal of Medical Virology, ISSN 0146-6615, E-ISSN 1096-9071, Vol. 87, no 7, p. 1130-1140Article in journal (Refereed)
    Abstract [en]

    Exposure to Ljungan virus (LV) is implicated in the risk of autoimmune (type 1) diabetes but possible contribution by other parechoviruses is not ruled out. The aim was to compare children diagnosed with type 1 diabetes in 2005-2011 (n=69) with healthy controls (n=294), all from the Jamtland County in Sweden, using an exploratory suspension multiplex immunoassay for IgM and IgG against 26 peptides of LV, human parechoviruses (HPeV), Aichi virus and poliovirus in relation to a radiobinding assay (RBA) for antibodies against LV and InfluenzaA/H1N1pdm09. Islet autoantibodies and HLA-DQ genotypes were also determined. 1) All five LV-peptide antibodies correlated to each other (P<0.001) in the suspension multiplex IgM- and IgG-antibody assay; 2) The LV-VP1_31-60-IgG correlated with insulin autoantibodies alone (P=0.007) and in combination with HLA-DQ8 overall (P=0.022) as well as with HLA-DQ 8/8 and 8/X subjects (P=0.013); 3) RBA detected LV antibodies correlated with young age at diagnosis (P<0.001) and with insulin autoantibodies (P<0.001) especially in young HLA-DQ8 subjects (P=0.004); 4) LV-peptide-VP1_31-60-IgG correlated to RBA LV antibodies (P=0.009); 5) HPeV3-peptide-IgM and -IgG showed inter-peptide correlations (P<0.001) but only HPeV3-VP1_1-30-IgG (P<0.001) and VP1_95-124-IgG (P=0.009) were related to RBA LV antibodies without relation to insulin autoantibody positivity (P=0.072 and P=0.486, respectively). Both exploratory suspension multiplex IgG to LV-peptide VP1_31-60 and RBA detected LV antibodies correlated with insulin autoantibodies and HLA-DQ8 suggesting possible role in type 1 diabetes. It remains to be determined if cross-reactivity or concomitant exposure to LV and HPeV3 contributes to the seroprevalence. J. Med. Virol. 87:1130-1140, 2015.

  • 55.
    Oja, Merja
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Sciences.
    Sperber, Göran
    Department of Neuroscience.
    Blomberg, Jonas
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Sciences.
    Kaski, Samuel
    Self-organizing map-based discovery and visualization of human endogenous retroviral2005In: Int J Neural Syst, ISSN 0129-0657, Vol. 15, no 3, p. 163-79Article in journal (Refereed)
  • 56.
    Pisano, Maria Paola
    et al.
    Univ Cagliari, Dept Life & Environm Sci, Lab Mol Virol, Cagliari, Italy.
    Grandi, Nicole
    Univ Cagliari, Dept Life & Environm Sci, Lab Mol Virol, Cagliari, Italy.
    Cadeddu, Marta
    Univ Cagliari, Dept Life & Environm Sci, Lab Mol Virol, Cagliari, Italy.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Tramontano, Enzo
    Univ Cagliari, Dept Life & Environm Sci, Lab Mol Virol, Cagliari, Italy;CNR, Ist Ric Genet & Biome, Cagliari, Italy.
    Comprehensive Characterization of the Human Endogenous Retrovirus HERV-K(HML-6) Group: Overview of Structure, Phylogeny, and Contribution to the Human Genome2019In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 93, no 16, article id e00110-19Article in journal (Refereed)
    Abstract [en]

    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. IMPORTANCE 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 env 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.

  • 57.
    Rönnberg, Bengt
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology.
    Gustafsson, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Univ Uppsala Hosp, Lab Clin Microbiol, Uppsala, Sweden..
    Vapalahti, Olli
    Univ Helsinki, Dept Vet Biosci & Virol, Helsinki, Finland.;Helsinki Univ Hosp, Helsinki, Finland..
    Emmerich, Petra
    Bernhard Nocht Inst Trop Med, WHO Collaborating Ctr Arbovirus & Haemorrhag Feve, D-20359 Hamburg, Germany.;Univ Rostock, Dept Trop Med & Infect Dis, Ctr Internal Med 2, D-18057 Rostock, Germany..
    Lundkvist, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology. Univ Uppsala Hosp, Lab Clin Microbiol, Uppsala, Sweden..
    Schmidt-Chanasit, Jonas
    Bernhard Nocht Inst Trop Med, WHO Collaborating Ctr Arbovirus & Haemorrhag Feve, D-20359 Hamburg, Germany.;German Ctr Infect Res DZIF, Partner Site Hamburg Luebeck Borstel, Hamburg, Germany..
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology.
    Compensating for cross-reactions using avidity and computation in a suspension multiplex immunoassay for serotyping of Zika versus other flavivirus infections2017In: Medical Microbiology and Immmunology, ISSN 0300-8584, E-ISSN 1432-1831, Vol. 206, no 5, p. 383-401Article in journal (Refereed)
    Abstract [en]

    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.

  • 58.
    Schmidt, Peter
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Forsman, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Andersson, Göran
    Blomberg, Jonas
    Korsgren, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Pig islet xenotransplantation: activation of porcine endogenous retrovirus in the immediate post-transplantation period2005In: Xenotransplantation, ISSN 0908-665X, E-ISSN 1399-3089, Vol. 12, no 6, p. 450-456Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Porcine endogenous retroviruses (PERV) are considered as the main infectious barrier in islet xenotransplantation. PERV has been shown to infect, but not to cause symptomatic disease in mice after islet transplantation. In vivo activation of PERV have so far not been examined. Expression of PERV was examined in adult and fetal porcine islets with or without the presence of known retroviral inducers or after transplantation to rats.

    METHODS: Isolated adult and fetal porcine islets were cultured under normal conditions or in the presence of dexamethasone or 5-azacytidine and 5-iodo-2-deoxyuridine. PERV mRNA content was analyzed by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and culture supernatants were analyzed for the presence of retroviral RT. Also, fetal islets were transplanted under the kidney capsule of immunocompetent or nude athymic rats. Expression of PERV mRNA in the grafts was evaluated by real-time quantitative RT-PCR. Infiltration of immunocompetent cells were evaluated by immunohistochemistry.

    RESULTS: Both fetal and adult islets in culture produced small or even undetectable amounts of PERV mRNA and retroviral RT. PERV expression was not enhanced by retroviral inducers. In contrast, activation of PERV expression was observed the first day after transplantation of fetal islet-like cell clusters in both athymic and normal rats. PERV expression peaked after 1 to 3 days and was then rapidly returned to background levels. PERV expression neither correlated with the innate immune response seen in athymic rats nor with the specific process of rejection in normal rats.

    CONCLUSION: Both fetal and adult islets produce low amounts of PERV mRNA in culture. After transplantation PERV expression is induced, seemingly independent of both the unspecific inflammatory response and the specific T-cell-mediated rejection process. It is speculated that PERV expression is correlated with the level of hypoxia in the islet xenograft.

  • 59. Shao, X-W
    et al.
    Hjalmarsson, Sandra
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Lennerstrand, Johan
    Division of Clinical Virology, Huddinge University Hospital.
    Svennerholm, B
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Källander, CFR
    Gronowitz, J Simon
    Application of a colorimetric chain-termination assay for characterization of reverse transcriptase from 3-azido-2,3-deoxythymidine-resistant HIV isolates2002In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 35, p. 155-164Article in journal (Refereed)
    Abstract [en]

    wo different enzyme assays, both based on the interaction of native reverse transcriptase(IRT) and 3'-azido-2',3'-deoxythymidine triphosphate (AZT-TP), were used to characterize the enzymesfrom 18 HIV-I isolates with decreased sensitivity to AZT in cell culture. The first assay, which measures the balance between incorporation and excision of AZT monophosphate in the presence of dNTP substrate (in terms of IC50), gave an approx. 9-fold variation in sensitivity to AZT-TP. There was a correlation between the IC50 values and the sensitivity of the corresponding virus to AZT in cell culture (r = 0.60, P < 0.01). The second assay, which was designed specifically for measurement of chain termination in the absence of dNTP substrate (as the concentration of AZT-TP giving 50% residual primer function, or CT50), revealed a more than 600-fold difference between the different isolate RTs. For the majority ofenzymes there was a strict correlation between the results from the two assays; however, four isolatesexhibited significantly higher CT50/IC50 ratios than the other isolates. These differences were not related to sensitivity of the corresponding viruses to AZT but to the occurrence of certain mutations in their pol gene. The four deviating isolates contained either a minimum of four AZT-specific substitutions, including Thr-215 --> Tyr (isolates 134 and 143), or some of the known specific substitutions combined with Thr-39 --> Ala (isolates 80 and 157). The Thr-39 Ala substitution has previously been recorded in connection with AZT/Foscarnet combination therapy.

  • 60. Sheikholvaezin, Ali
    et al.
    Blomberg, Fredrik
    Ohrmalm, Christina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Sjösten, Anna
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Rational recombinant XMRV antigen preparation and bead coupling for multiplex serology in a suspension array2011In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 80, no 2, p. 176-184Article in journal (Refereed)
    Abstract [en]

    Diagnosis of infectious diseases often requires demonstration of antibodies to the microbe (serology). A large set of antigens, covering viruses, bacteria, fungi and parasites may be needed. Recombinant proteins have a prime role in serological tests. Suspension arrays offer high throughput for simultaneous measurement of many different antibodies. We here describe a rational process for preparation, purification and coupling to beads of recombinant proteins prepared in Escherichia coli derivate Origami B, to be used in a serological Luminex suspension array. All six Gag and Env proteins (p10, p12, p15, p30, gp70 and p15E), from the xenotropic murine leukemia virus-related virus (XMRV), were prepared, allowing the creation of a multiepitope XMRV antibody assay. The procedure is generic and allows production of protein antigens ready for serological testing in a few working days. Instability and aggregation problems were circumvented by expression of viral proteins fused to a carrier protein (thioredoxin A; TrxA), purification via inclusion body formation, urea solubilization, His tag affinity chromatography and direct covalent coupling to microspheres without removal of the elution buffer. The yield of one preparation (2-10 mg fusion protein per 100 ml culture) was enough for 20-100 coupling reactions, sufficing for tests of many tens of thousands of sera. False serological positivity due to antibodies binding to TrxA and to traces of E. coil proteins remaining in the preparation could be reduced by preabsorption of sera with free TrxA and E. coli extract. The recombinant antigens were evaluated using anti-XMRV antibodies. Although hybrid proteins expressed in E. coli in this way will not have the entire tertiary structure and posttranslational modifications of the native proteins, they contain a large subset of the epitopes associated with them. The described strategy is simple, quick, efficient and cheap. It should be applicable for suspension array serology in general.

  • 61.
    Sperber, Göran
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Lövgren, Anders
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Eriksson, Nils-Einar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Benachenhou, Farid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    RetroTector online, a rational tool for analysis of retroviral elements in small and medium size vertebrate genomic sequences2009In: BMC Bioinformatics, ISSN 1471-2105, E-ISSN 1471-2105, Vol. 10 Suppl 6, p. S4-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: The rapid accumulation of genomic information in databases necessitates rapid and specific algorithms for extracting biologically meaningful information. More or less complete retroviral sequences, also called proviral or endogenous retroviral sequences; ERVs, constitutes at least 5% of vertebrate genomes. After infecting the host, these retroviruses have integrated in germ line cells, and have then been carried in genomes for at least several 100 million years. A better understanding of structure and function of these sequences can have profound biological and medical consequences. METHODS: RetroTector (ReTe) is a platform-independent Java program for identification and characterization of proviral sequences in vertebrate genomes. The full ReTe requires a local installation with a MySQL database. Although not overly complicated, the installation may take some time. A "light" version of ReTe, (RetroTector online; ROL) which does not require specific installation procedures is provided, via the World Wide Web. RESULT: ROL http://www.fysiologi.neuro.uu.se/jbgs/ was implemented under the Batchelor web interface (A Lövgren et al). It allows both GenBank accession number, file and FASTA cut-and-paste admission of sequences (5 to 10,000 kilobases). Up to ten submissions can be done simultaneously, allowing batch analysis of <or= 100 Megabases. Jobs are shown in an IP-number specific list. Results are text files, and can be viewed with the program, RetroTectorViewer.jar (at the same site), which has the full graphical capabilities of the basic ReTe program. A detailed analysis of any retroviral sequences found in the submitted sequence is graphically presented, exportable in standard formats. With the current server, a complete analysis of a 1 Megabase sequence is complete in 10 minutes. It is possible to mask nonretroviral repetitive sequences in the submitted sequence, using host genome specific "brooms", which increase specificity. DISCUSSION: Proviral sequences can be hard to recognize, especially if the integration occurred many million years ago. Precise delineation of LTR, gag, pro, pol and env can be difficult, requiring manual work. ROL is a way of simplifying these tasks. CONCLUSION: ROL provides 1. annotation and presentation of known retroviral sequences, 2. detection of proviral chains in unknown genomic sequences, with up to 100 Mbase per submission.

  • 62.
    Sperber, Göran O.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Airola, Tove
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Jern, Patric
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Automated recognition of retroviral sequences in genomic data - RetroTector©2007In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 35, no 15, p. 4964-4976Article in journal (Refereed)
    Abstract [en]

    Eukaryotic genomes contain many endogenous retroviral sequences(ERVs). ERVs are often severely mutated, therefore difficultto detect. A platform independent (Java) program package, RetroTector©(ReTe), was constructed. It has three basic modules: (i) detectionof candidate long terminal repeats (LTRs), (ii) detection ofchains of conserved retroviral motifs fulfilling distance constraintsand (iii) attempted reconstruction of original retroviral proteinsequences, combining alignment, codon statistics and propertiesof protein ends. Other features are prediction of additionalopen reading frames, automated database collection, graphicalpresentation and automatic classification. ReTe favors elements>1000-bp long due to its dependence on order of and distancesbetween retroviral fragments. It detects single or low-copy-numberelements. ReTe assigned a ‘retroviral’ score of890–2827 to 10 exogenous retroviruses from seven genera,and accurately predicted their genes. In a simulated model,ReTe was robust against mutational decay. The human genome wasanalyzed in 1–2 days on a LINUX cluster. Retroviral sequenceswere detected in divergent vertebrate genomes. Most ReTe detectedchains were coincident with Repeatmasker output and the HERVddatabase. ReTe did not report most of the evolutionary old HERV-Lrelated and MalR sequences, and is not yet tailored for singleLTR detection. Nevertheless, ReTe rationally detects and annotatesmany retroviral sequences.

  • 63.
    Vargiu, Laura
    et al.
    Univ Cagliari, Dept Life & Environm Sci, Cagliari, Italy.;CRS4, Ctr Adv Studies Res & Dev Sardinia, Pula, Italy.;Nurideas Srl, Cagliari, Italy..
    Rodriguez-Tome, Patricia
    CRS4, Ctr Adv Studies Res & Dev Sardinia, Pula, Italy.;Nurideas Srl, Cagliari, Italy..
    Sperber, Göran O.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Cadeddu, Marta
    Univ Cagliari, Dept Life & Environm Sci, Cagliari, Italy..
    Grandi, Nicole
    Univ Cagliari, Dept Life & Environm Sci, Cagliari, Italy..
    Blikstad, Vidar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine.
    Tramontano, Enzo
    Univ Cagliari, Dept Life & Environm Sci, Cagliari, Italy..
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine.
    Classification and characterization of human endogenous retroviruses: mosaic forms are common2016In: Retrovirology, ISSN 1742-4690, E-ISSN 1742-4690, Vol. 13, article id 7Article in journal (Refereed)
    Abstract [en]

    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.

  • 64. Vargiu, Luana
    et al.
    Rodriguez-Tome, Patricia
    Sperber, Göran O.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Tramontano, Enzo
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Overview of human endogenous retroviruses found in human genome assembly GRCh37/hg192013In: Retrovirology, ISSN 1742-4690, E-ISSN 1742-4690, Vol. 10, no S1, p. P6-Article in journal (Other academic)
  • 65.
    Wang, Yilin
    et al.
    Univ Helsinki, Virol, FI-00290 Helsinki, Finland..
    Hedman, Lea
    Helsinki Univ Hosp, Helsinki, Finland..
    Perdomo, Maria F.
    Univ Helsinki, Virol, FI-00290 Helsinki, Finland..
    Elfaitouri, Amal
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Bolin-Wiener, Agnes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Kumar, Arun
    Univ Helsinki, Virol, FI-00290 Helsinki, Finland..
    Lappalainen, Maija
    Univ Helsinki, Virol, FI-00290 Helsinki, Finland.;Helsinki Univ Hosp, Helsinki, Finland..
    Soderlund-Venermo, Maria
    Univ Helsinki, Virol, FI-00290 Helsinki, Finland..
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine.
    Hedman, Klaus
    Univ Helsinki, Virol, FI-00290 Helsinki, Finland.;Helsinki Univ Hosp, Helsinki, Finland..
    Microsphere-based antibody assays for human parvovirus B19V, CMV and T. gondii2016In: BMC Infectious Diseases, ISSN 1471-2334, E-ISSN 1471-2334, Vol. 16, article id 8Article in journal (Refereed)
    Abstract [en]

    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.

  • 66.
    Westman, Gabriel
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine.
    Yun, Zhibing
    Karolinska Inst, Dept Lab Med, Stockholm, Sweden..
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Ingelsson, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Eriksson, Britt-Marie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Decreased HHV-6 IgG in Alzheimer's Disease2017In: Frontiers in Neurology, ISSN 1664-2295, E-ISSN 1664-2295, Vol. 8, article id 40Article in journal (Refereed)
    Abstract [en]

    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.

  • 67.
    Xia, Hongyan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine. Swedish Univ Agr Sci, Dept Biomed Sci & Vet Publ Hlth, S-75007 Uppsala, Sweden..
    Gravelsina, Sabine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine. Riga Stradins Univ, August Kirchenstein Inst Microbiol & Virol, Riga, Latvia..
    Öhrmalm, Christina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology.
    Ottoson, Jakob
    Swedish Univ Agr Sci, Dept Biomed Sci & Vet Publ Hlth, S-75007 Uppsala, Sweden.;Natl Food Adm Toxicol Lab, Dept Risk & Benefit Assessment, Uppsala, Sweden..
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Development of single-tube nested real-time PCR assays with long internally quenched probes for detection of norovirus genogroup II2016In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 60, no 1, p. 28-34Article in journal (Refereed)
    Abstract [en]

    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.

  • 68.
    Öhrmalm, Christina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Eriksson, Ronnie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Jobs, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Simonson, Magnus
    Naitonal Food Agency, Uppsala.
    Strømme, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Nanotechnology and Functional Materials.
    Bondeson, Kåre
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Herrmann, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Bacteriology.
    Melhus, Åsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Bacteriology.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Variation-tolerant capture and multiplex detection of nucleic acids: application to detection of microbes2012In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 50, no 10, p. 3208-3215Article in journal (Refereed)
    Abstract [en]

    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.

  • 69.
    Öhrmalm, Christina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Jobs, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Eriksson, Ronnie
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Golbob, Sultan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Elfaitouri, Amal
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Benachenhou, Farid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Strømme, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Nanotechnology and Functional Materials.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Hybridization properties of long nucleic acid probes for detection of variable target sequences, and development of a hybridization prediction algorithm2010In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 21, p. e195-Article in journal (Refereed)
    Abstract [en]

    One of the main problems in nucleic acid-based techniques for detection of infectious agents, such as influenza viruses, is that of nucleic acid sequence variation. DNA probes, 70-nt long, some including the nucleotide analog deoxyribose-Inosine (dInosine), were analyzed for hybridization tolerance to different amounts and distributions of mismatching bases, e.g. synonymous mutations, in target DNA. Microsphere-linked 70-mer probes were hybridized in 3M TMAC buffer to biotinylated single-stranded (ss) DNA for subsequent analysis in a Luminex® system. When mismatches interrupted contiguous matching stretches of 6 nt or longer, it had a strong impact on hybridization. Contiguous matching stretches are more important than the same number of matching nucleotides separated by mismatches into several regions. dInosine, but not 5-nitroindole, substitutions at mismatching positions stabilized hybridization remarkably well, comparable to N (4-fold) wobbles in the same positions. In contrast to shorter probes, 70-nt probes with judiciously placed dInosine substitutions and/or wobble positions were remarkably mismatch tolerant, with preserved specificity. An algorithm, NucZip, was constructed to model the nucleation and zipping phases of hybridization, integrating both local and distant binding contributions. It predicted hybridization more exactly than previous algorithms, and has the potential to guide the design of variation-tolerant yet specific probes.

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