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  • 1.
    Abdeldaim, Guma M. K.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Bacteriology.
    Stralin, Kristoffer
    Olcen, Per
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Molling, Paula
    Herrmann, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Bacteriology.
    Quantitative fucK gene polymerase chain reaction on sputum and nasopharyngeal secretions to detect Haemophilus influenzae pneumonia2013In: Diagnostic microbiology and infectious disease, ISSN 0732-8893, E-ISSN 1879-0070, Vol. 76, no 2, p. 141-146Article in journal (Refereed)
    Abstract [en]

    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.

  • 2.
    Abdeldaim, Guma
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Bacteriology. Benghazi Univ, Fac Med, Dept Med Microbiol & Parasitol, Benghazi, Libya..
    Svensson, Erik
    Statens Serum Inst, Int Reference Lab Mycobacteriol, Copenhagen, Denmark..
    Blomberg, Jonas
    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.
    Duplex detection of the Mycobacterium tuberculosis complex and medically important non-tuberculosis mycobacteria by real-time PCR based on the rnpB gene2016In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 124, no 11, p. 991-995Article in journal (Refereed)
    Abstract [en]

    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 nonrespiratory 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.

  • 3.
    Benachenhou, Farid
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Sperber, Göran O.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Bongcam-Rudloff, Erik
    Andersson, Goran
    Boeke, Jef D.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Conserved structure and inferred evolutionary history of long terminal repeats (LTRs)2013In: Mobile DNA, ISSN 1759-8753, E-ISSN 1759-8753, Vol. 4, p. 5-Article in journal (Refereed)
    Abstract [en]

    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.

  • 4.
    Bergfors, Assar
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine.
    Leenheer, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology. Univ Tsukuba, PhD Program Human Biol, Sch Integrat & Global Majors, Tsukuba, Ibaraki 3058577, Japan..
    Bergqvist, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine.
    Ameur, Adam
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Lennerstrand, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Analysis of hepatitis C NS5A resistance associated polymorphisms using ultra deep single molecule real time (SMRT) sequencing2016In: Antiviral Research, ISSN 0166-3542, E-ISSN 1872-9096, Vol. 126, p. 81-89Article in journal (Refereed)
    Abstract [en]

    Development of Hepatitis C virus (HCV) resistance against direct-acting antivirals (DAAs), including NS5A inhibitors, is an obstacle to successful treatment of HCV when DAAs are used in sub-optimal combinations. Furthermore, it has been shown that baseline (pre-existing) resistance against DAAs is present in treatment naive-patients and this will potentially complicate future treatment strategies in different HCV genotypes (GTs). Thus the aim was to detect low levels of NS5A resistant associated variants (RAVs) in a limited sample set of treatment-naive patients of HCV GT1a and 3a, since such polymorphisms can display in vitro resistance as high as 60000 fold. Ultra-deep single molecule real time (SMRT) sequencing with the Pacific Biosciences (PacBio) RSII instrument was used to detect these RAVs. The SMRT sequencing was conducted on ten samples; three of them positive with Sanger sequencing (GT1a Q30H and Y93N, and GT3a Y93H), five GT1a samples, and two GT3a non-positive samples. The same methods were applied to the HCV GT1a H77-plasmid in a dilution series, in order to determine the error rates of replication, which in turn was used to determine the limit of detection (LOD), as defined by mean + 3SD, of minority variants down to 0.24%. We found important baseline NS5A RAVs at levels between 0.24 and 0.5%, which could potentially have clinical relevance. This new method with low level detection of baseline RAVs could be useful in predicting the most cost-efficient combination of DAA treatment, and reduce the treatment duration for an HCV infected individual.

  • 5. Bjohle, J.
    et al.
    Bergqvist, J.
    Gronowitz, J. Simon
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Johansson, H.
    Carlsson, L.
    Einbeigi, Z.
    Linderholm, B.
    Loman, N.
    Malmberg, M.
    Soderberg, M.
    Sundquist, M.
    Walz, T. M.
    Ferno, M.
    Bergh, J.
    Hatschek, T.
    Serum thymidine kinase activity compared with CA 15-3 in locally advanced and metastatic breast cancer within a randomized trial2013In: Breast Cancer Research and Treatment, ISSN 0167-6806, E-ISSN 1573-7217, Vol. 139, no 3, p. 751-758Article in journal (Refereed)
    Abstract [en]

    The primary objective was to estimate serum thymidine kinase 1 (TK1) activity, reflecting total body cell proliferation rate including cancer cell proliferation, in women with loco regional inoperable or metastatic breast cancer participating in a prospective and randomized study. Secondary objectives were to analyze TK1 in relation to progression-free survival (PFS), overall survival (OS), therapy response and other tumour characteristics, including CA 15-3, widely used as a standard serum marker for disease progression. TK1 and CA 15-3 were analysed in 198 serum samples collected prospectively from women included in the randomized TEX trial between December 2002 and June 2007. TK1 activity was determined by the ELISA based DiviTum (TM) assay, and CA 15-3 analyses was generated with the electrochemiluminescence immunoassay Cobas Elecsys CA 15-3 II. High pre-treatment TK1 activity predicted shorter PFS (10 vs. 15 months p = 0.02) and OS (21 vs. 38 months, p < 0.0001), respectively. After adjustment for age, metastatic site and study treatment TK1 showed a trend as predictor of PFS (p = 0.059) and was an independent prognostic factor for OS, (HR 1.81, 95 % confidence interval (CI) 1.26-2.61, p = 0.001). There was a trend of shortened OS for women with high CA 15-3 (p = 0.054) in univariate analysis, but not after adjustment for the above mentioned covariates. Both TK1 (p = 0.0011) and CA 15-3 (p = 0.0004) predicted response to treatment. There were statistically different distributions of TK1 and CA 15-3 in relation to the site of metastases. TK1 activity measured by DiviTum (TM) predicted therapy response, PFS and OS in loco regional inoperable or disseminated breast cancer. These results suggest that this factor is a useful serum marker. In the present material, a prognostic value of CA 15-3 could not be proven.

  • 6.
    Blomberg, Jonas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Blomberg, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Sjösten, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Sheikholvaezin, Ali
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Bölin-Wiener, Agnes
    Elfaitouri, Amal
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Hessel, Sanna
    Gottfries, Carl-Gerhard
    Zachrisson, Olof
    Öhrmalm, Christina
    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.
    Pipkorn, Ruediger
    No Evidence for Xenotropic Murine Leukemia-Related Virus Infection in Sweden Using Internally Controlled Multiepitope Suspension Array Serology2012In: Clinical and Vaccine Immunology, ISSN 1556-6811, E-ISSN 1556-679X, Vol. 19, no 9, p. 1399-1410Article in journal (Refereed)
    Abstract [en]

    Many syndromes have a large number of differential diagnoses, a situation which calls for multiplex diagnostic systems. Myalgic encephalomyelitis (ME), also named chronic fatigue syndrome (CFS), is a common disease of unknown etiology. A mouse retrovirus, xenotropic murine leukemia-related virus (XMRV), was found in ME/CFS patients and blood donors, but this was not corroborated. However, the paucity of serological investigations on XMRV in humans prompted us to develop a serological assay which cover many aspects of XMRV antigenicity. It is a novel suspension array method, using a multiplex IgG assay with nine recombinant proteins from the env and gag genes of XMRV and 38 peptides based on known epitopes of vertebrate gamma-retroviruses. IgG antibodies were sought in 520 blood donors and 85 ME/CFS patients and in positive-and negative-control sera from animals. We found no differences in seroreactivity between blood donors and ME/CFS patients for any of the antigens. This did not support an association between ME/CFS and XMRV infection. The multiplex serological system had several advantages: (i) biotinylated protein G allowed us to run both human and animal sera, which is essential because of a lack of XMRV-positive humans; (ii) a novel quality control was a pan-peptide positive-control rabbit serum; and (iii) synthetic XMRV Gag peptides with degenerate positions covering most of the variation of murine leukemia-like viruses did not give higher background than nondegenerate analogs. The principle may be used for creation of variant tolerant peptide serologies. Thus, our system allows rational large-scale serological assays with built-in quality control.

  • 7. Bolisetty, M.
    et al.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Benachenhou, Farid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Sperber, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Beemon, K.
    Unexpected diversity and expression of avian endogenous retroviruses2012In: mBio, ISSN 2161-2129, Vol. 3, no 5, p. e00344-12Article in journal (Refereed)
    Abstract [en]

    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.

  • 8. Cadeddu, Marta
    et al.
    Vargiu, Laura
    Rodriguez-Tome, Patricia
    Sperber, Göran O.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Tramontano, Enzo
    Identification and analysis of HML2 sequences in human genome assembly GRCh37/hg192013In: Retrovirology, ISSN 1742-4690, E-ISSN 1742-4690, Vol. 10, no S1, p. P9-Article in journal (Other academic)
  • 9. Camussone, C. M.
    et al.
    Pujato, N.
    Renna, M. S.
    Veaute, C. M.
    Morein, Bror
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Marcipar, I. S.
    Calvinho, L. F.
    Immune response and functional role of antibodies raised in heifers against a Staphylococcus aureus CP5 lysate and recombinant antigens vaccine formulated with Iscom Matrix adjuvant2014In: Veterinary Immunology and Immunopathology, ISSN 0165-2427, E-ISSN 1873-2534, Vol. 162, no 3-4, p. 96-107Article in journal (Refereed)
    Abstract [en]

    Staphylococcus aureus is the most frequently isolated pathogen from bovine intramammary infections worldwide. Commercially available vaccines for mastitis control are composed either of S. aureus lysates or inactivated whole-cells formulated with traditional adjuvants. We recently showed the ability of a S. aureus CP5 lysate vaccine adjuvanted with Iscom Matrix to generate a longer lasting specific antibody response in blood and milk with improved opsonic capacity compared with a S. aureus CP5 whole-cell formulation. The aim of the present study was to obtain an experimental immunogen composed of lysed cells of a CP5 S. aureus strain supplemented with recombinant clumping factor A fibronectin binding protein A and beta-toxin formulated with Iscom Matrix characterize the immune response generated when immunizing pregnant heifers and assess the functional role of antibodies raised against this immunogen in experimental models. Both a lysate vaccine and a lysate + recombinant antigens vaccine elicited antibodies that promoted neutrophil phagocytosis and inhibited internalization into mammary epithelial cells in vitro. Incorporation of defined antigenic molecules to the lysate formulation elicited a strong specific humoral immune response against both lysate and recombinant antigens and was associated with higher expression of regulatory and pro-inflammatory cytokines. In addition antibodies were efficient for blocking S. aureus binding to bovine fibrinogen and fibronectin and neutralizing beta-toxin effect in vitro placing these antigens as candidates to be included in a formulation directed to prevent staphylococcal bovine mastitis. (C) 2014 Elsevier B.V. All rights reserved.

  • 10. Camussone, C. M.
    et al.
    Veaute, C. M.
    Porporatto, C.
    Morein, Bror
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Marcipar, I. S.
    Calvinho, L. F.
    Immune response of heifers against a Staphylococcus aureus CP5 whole cell vaccine formulated with ISCOMATRIX™ adjuvant2013In: Journal of dairy research (Print), ISSN 0022-0299, E-ISSN 1469-7629, Vol. 80, no 1, p. 72-80Article in journal (Refereed)
    Abstract [en]

    The shortcomings of Staphylococcus aureus vaccines to control bovine mastitis have been attributed to insufficient capacity of the vaccines to induce opsonizing antibodies and to stimulate cellular immune responses. Types of antigen, administration route and adjuvant used in a vaccine formulation have been identified as critical factors for the development of opsonic antibodies. Current commercially available vaccines for Staph. aureus bovine mastitis control are formulated with Al(OH)3 and oil-based adjuvants. The aim of this study was to evaluate the immune response of heifers immunized with a Staph. aureus CP5 whole cell vaccine formulated either with Al(OH)3 or ISCOMATRIX ™. Twenty primigravid Holstein dairy heifers in the last trimester of gestation were immunized either with a vaccine formulated with ISCOMATRIX™ (n = 6), Al(OH)3 (n = 7), or saline solution (placebo) (n = 7). Immunization was carried out 38 and 10 d before calving. Heifers vaccinated with Staph. aureus adjuvanted with ISCOMATRIX™ responded with significantly higher levels of anti-bacterin and anti-CP5 IgG and IgG2 in sera than animals in the Al(OH)3 or control groups. Animals in the ISCOMATRIX™ group responded with significantly higher anti-bacterin specific IgG in whey than animals in the Al(OH)3 and control groups, detected from the first week post calving until 60 d of lactation. Sera from animals inoculated with Staph. aureus in ISCOMATRIX™, obtained 7 d post partum, significantly increased both the number of neutrophils ingesting bacteria and the number of bacteria being ingested by the neutrophils, compared with sera obtained from heifers vaccinated with Al(OH)3 or non-vaccinated controls. These features coupled to safety of the ISCOMATRIX™ formulation, warrant additional studies.

  • 11. Camussone, Cecilia M.
    et al.
    Veaute, Carolina M.
    Pujato, Nazarena
    Morein, Bror
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Marcipar, Ivan S.
    Calvinho, Luis F.
    Immune response of heifers against a Staphylococcus aureus CP5 whole cell and lysate vaccine formulated with ISCOM Matrix adjuvant2014In: Research in Veterinary Science, ISSN 0034-5288, E-ISSN 1532-2661, Vol. 96, no 1, p. 86-94Article in journal (Refereed)
    Abstract [en]

    Staphylococcus aureus is the most frequently isolated pathogen from bovine intramammary infections worldwide. Commercially available vaccines for mastitis control are composed either of S. aureus lysates or whole-cells formulated with traditional adjuvants. We recently showed the ability of a S. aureus CP5 whole-cell vaccine adjuvanted with ISCOM Matrix to increase specific antibodies production in blood and milk, improving opsonic capacity, compared with the same vaccine formulated with Al(OH)(3). However, there is no information about the use of ISCOM Matrix for the formulation of bacterial lysates. The aim of this study was to characterize the innate and humoral immune responses induced by a S. aureus CP5 whole-cell or lysate vaccine, formulated with ISCOM Matrix after immunization of pregnant heifers. Both immunogens stimulated strong humoral immune responses in blood and milk, raising antibodies that increased opsonic capacity. Lysate formulation generated a higher and longer lasting antibody titer and stimulated a higher expression of regulatory and pro-inflammatory cytokines compared with the whole-cell vaccine. (C) 2013 Elsevier Ltd. All rights reserved.

  • 12.
    Danielsson, Axel
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Palanisamy, Navaneethan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Golbob, Sultan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Yin, Hong
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Hedlund, Johan
    Sylvan, Staffan
    Lennerstrand, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Transmission of hepatitis C virus among intravenous drug users in the Uppsala region of Sweden2014In: Infection Ecology & Epidemiology, ISSN 2000-8686, E-ISSN 2000-8686, Vol. 4, article id 22251Article in journal (Refereed)
    Abstract [en]

    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).

  • 13. Groenen, M. A.
    et al.
    Archibald, A. L.
    Uenishi, H.
    Tuggle, C. K.
    Takeuchi, Y.
    Rothschild, M. F.
    Rogel-Gaillard, C.
    Park, C.
    Milan, D.
    Megens, H. J.
    Li, S.
    Larkin, D. M.
    Kim, H.
    Frantz, L. A.
    Caccamo, M.
    Ahn, H.
    Aken, B. L.
    Anselmo, A.
    Anthon, C.
    Auvil, L.
    Badaoui, B.
    Beattie, C. W.
    Bendixen, C.
    Berman, D.
    Blecha, F.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Bolund, L.
    Bosse, M.
    Botti, S.
    Bujie, Z.
    Byström, M.
    Capitanu, B.
    Carvalho-Silva, D.
    Chardon, P.
    Chen, C.
    Cheng, R.
    Choi, S. H.
    Chow, W.
    Clark, R. C.
    Clee, C.
    Crooijmans, R. P.
    Dawson, H. D.
    Dehais, P.
    De Sapio, F.
    Dibbits, B.
    Drou, N.
    Du, Z. Q.
    Eversole, K.
    Fadista, J.
    Fairley, S.
    Faraut, T.
    Faulkner, G. J.
    Fowler, K. E.
    Fredholm, M.
    Fritz, E.
    Gilbert, J. G.
    Giuffra, E.
    Gorodkin, J.
    Griffin, D. K.
    Harrow, J. L.
    Hayward, Alexander
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Howe, K.
    Hu, Z. L.
    Humphray, S. J.
    Hunt, T.
    Hornshoj, H.
    Jeon, J. T.
    Jern, Patric
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Jones, M.
    Jurka, J.
    Kanamori, H.
    Kapetanovic, R.
    Kim, J.
    Kim, J. H.
    Kim, K. W.
    Kim, T. H.
    Larson, G.
    Lee, K.
    Lee, K. T.
    Leggett, R.
    Lewin, H. A.
    Li, Y.
    Liu, W.
    Loveland, J. E.
    Lu, Y.
    Lunney, J. K.
    Ma, J.
    Madsen, O.
    Mann, K.
    Matthews, L.
    McLaren, S.
    Morozumi, T.
    Murtaugh, M. P.
    Narayan, J.
    Nguyen, D. T.
    Ni, P.
    Oh, S. J.
    Onteru, S.
    Panitz, F.
    Park, E. W.
    Park, H. S.
    Pascal, G.
    Paudel, Y.
    Perez-Enciso, M.
    Ramirez-Gonzalez, R.
    Reecy, J. M.
    Rodriguez-Zas, S.
    Rohrer, G. A.
    Rund, L.
    Sang, Y.
    Schachtschneider, K.
    Schraiber, J. G.
    Schwartz, J.
    Scobie, L.
    Scott, C.
    Searle, S.
    Servin, B.
    Southey, B. R.
    Sperber, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Physiology.
    Stadler, P.
    Sweedler, J. V.
    Tafer, H.
    Thomsen, B.
    Wali, R.
    Wang, J.
    White, S.
    Xu, X.
    Yerle, M.
    Zhang, G.
    Zhang, J.
    Zhao, S.
    Rogers, J.
    Churcher, C.
    Schook, L. B.
    Analyses of pig genomes provide insight into porcine demography and evolution2012In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 491, no 7424, p. 393-398Article in journal (Refereed)
    Abstract [en]

    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 approximately 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.

  • 14.
    Gronowitz, Simon J.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Nisman, B.
    Peretz-Yablonski, T.
    Total cell division the ultimate biomarker for personalized medicine in cancer?: updated technology and novel clinical results2012In: Annals of Oncology, ISSN 0923-7534, E-ISSN 1569-8041, Vol. 23, no S9, p. 88-88Article in journal (Other academic)
  • 15.
    Gronowitz, Simon
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Nisman, B.
    Peretz, Tamar
    Total body cell division the ultimate biomarker for personalized medicine in cancer?2012In: Tumor Biology, ISSN 1010-4283, E-ISSN 1423-0380, Vol. 33, no S1, p. 107-107Article in journal (Other academic)
  • 16.
    Hu, Lijuan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Andersson, G.
    Jonsson, Kenneth B.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics.
    Melhus, Håkan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical pharmacogenomics and osteoporosis.
    Lind, Thomas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical pharmacogenomics and osteoporosis.
    Adamts1 is highly induced in rachitic bones of FGF23 transgenic mice and participates in degradation of non-mineralized bone matrix collagen2013In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 430, no 3, p. 901-906Article in journal (Refereed)
    Abstract [en]

    Transgenic mice overexpressing fibroblast growth factor 23 (FGF23) in osteoblasts have a rachitic bone phenotype. These mice display hypomineralized bones, increased expression of osteoblast markers, but osteoclast numbers are unaltered or slightly reduced. Paradoxically, they show increased serum levels of the bone resorption marker CTX, a type I collagen degradation fragment. Here we analyzed a matrix metalloproteinase- (MMP-) like secreted protease, Adamts1, that has previously been associated with osteoblastic type I collagen breakdown in vitro. Bones from FGF23 transgenic (tg) mice displayed increased Adamts1 protein upon both immunohistological staining and Western blotting. We further found Adamts1 protein together with excessively degraded type I collagen in the non-mineralized bone fraction of FGF23 tg mice. A similar degradation pattern of type I collagen was noticed upon forced expression of Adamts1 in osteoblastic cells in vitro. Importantly, these Adamts1-expressing osteoblastic cells exhibited increased release of CTX fragments when cultured on demineralized bone discs. Together, these results demonstrate for the first time that Adamts1 can be highly induced in bone tissue and that this MMP-like protease can increase osteoblastic release of CTX fragments from non-mineralized bone. Thus, Adamts1 potentially contributes to the increased serum levels of CTX in rickets/osteomalacia.

  • 17.
    Hu, Lijuan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Uzhameckis, Dmitrijs
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Hedborg, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Dynamic and selective HERV RNA expression in neuroblastoma cells subjected to variation in oxygen tension and demethylation2016In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 124, no 1-2, p. 140-149Article in journal (Refereed)
    Abstract [en]

    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.

  • 18.
    Järhult, Josef D.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Muradrasoli, Shaman
    Wahlgren, John
    Söderström, Hanna
    Orozovic, Goran
    Gunnarsson, Gunnar
    Bröjer, Caroline
    Latorre-Margalef, Neus
    Fick, Jerker
    Grabic, Roman
    Lennerstrand, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Waldenström, Jonas
    Lundkvist, Åke
    Olsen, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Environmental Levels of the Antiviral Oseltamivir Induce Development of Resistance Mutation H274Y in Influenza A/H1N1 Virus in Mallards2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 9, p. e24742-Article in journal (Refereed)
    Abstract [en]

    Oseltamivir (Tamiflu (R)) is the most widely used drug against influenza infections and is extensively stockpiled worldwide as part of pandemic preparedness plans. However, resistance is a growing problem and in 2008-2009, seasonal human influenza A/H1N1 virus strains in most parts of the world carried the mutation H274Y in the neuraminidase gene which causes resistance to the drug. The active metabolite of oseltamivir, oseltamivir carboxylate (OC), is poorly degraded in sewage treatment plants and surface water and has been detected in aquatic environments where the natural influenza reservoir, dabbling ducks, can be exposed to the substance. To assess if resistance can develop under these circumstances, we infected mallards with influenza A/H1N1 virus and exposed the birds to 80 ng/L, 1 mu g/L and 80 mu g/L of OC through their sole water source. By sequencing the neuraminidase gene from fecal samples, we found that H274Y occurred at 1 mu g/L of OC and rapidly dominated the viral population at 80 mu g/L. IC(50) for OC was increased from 2-4 nM in wild-type viruses to 400-700 nM in H274Y mutants as measured by a neuraminidase inhibition assay. This is consistent with the decrease in sensitivity to OC that has been noted among human clinical isolates carrying H274Y. Environmental OC levels have been measured to 58-293 ng/L during seasonal outbreaks and are expected to reach mu g/L-levels during pandemics. Thus, resistance could be induced in influenza viruses circulating among wild ducks. As influenza viruses can cross species barriers, oseltamivir resistance could spread to human-adapted strains with pandemic potential disabling oseltamivir, a cornerstone in pandemic preparedness planning. We propose surveillance in wild birds as a measure to understand the resistance situation in nature and to monitor it over time. Strategies to lower environmental levels of OC include improved sewage treatment and, more importantly, a prudent use of antivirals.

  • 19. Lindström, I.
    et al.
    Palanisamy, Navaneethan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Kjellin, M.
    Danielsson, Axel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Bondeson, Kåre
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Lennerstrand, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Implications of baseline polymorphisms for potential resistance to NS3 protease and NS5A inhibitors in hepatitis C virus genotypes 1a, 2b and 3a2013In: Antiviral Therapy, ISSN 1359-6535, E-ISSN 2040-2058, Vol. 18, no S1, p. A55-A55Article in journal (Other academic)
  • 20. Mayer, J.
    et al.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Seal, RL.
    A revised nomenclature for transcribed human endogenous retroviral loci2011In: Mobile DNA, ISSN 1759-8753, E-ISSN 1759-8753, Vol. 2, no 7Article in journal (Refereed)
    Abstract [en]

    Background

    Endogenous retroviruses (ERVs) and ERV-like sequences comprise 8% of the human genome. A hitherto unknown proportion of ERV loci are transcribed and thus contribute to the human transcriptome. A small proportion of these loci encode functional proteins. As the role of ERVs in normal and diseased biological processes is not yet established, transcribed ERV loci are of particular interest. As more transcribed ERV loci are likely to be identified in the near future, the development of a systematic nomenclature is important to ensure that all information on each locus can be easily retrieved.

    Results

    Here we present a revised nomenclature of transcribed human endogenous retroviral loci that sorts loci into groups based on Repbase classifications. Each symbol is of the format ERV + group symbol + unique number. Group symbols are based on a mixture of Repbase designations and well-supported symbols used in the literature. The presented guidelines will allow newly identified loci to be easily incorporated into the scheme.

    Conclusions

    The naming system will be employed by the HUGO Gene Nomenclature Committee for naming transcribed human ERV loci. We hope that the system will contribute to clarifying a certain aspect of a sometimes confusing nomenclature for human endogenous retroviruses. The presented system may also be employed for naming transcribed loci of human non-ERV repeat loci.

  • 21. 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.

  • 22. Nisman, B.
    et al.
    Kadouri, L.
    Allweis, T.
    Hamburger, T.
    Gronowitz, Simon
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Peretz, T.
    Proliferative background in healthy women carriers of BRCA1/2 mutation2012In: Tumor Biology, ISSN 1010-4283, E-ISSN 1423-0380, Vol. 33, no S1, p. 86-86Article in journal (Other academic)
  • 23. Nisman, B.
    et al.
    Nechushtan, H.
    Biran, H.
    Gronowitz, Simon
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Peretz, T.
    Evaluation of thymidine kinase 1 activity in the serum of lung cancer (LC) patients2012In: Tumor Biology, ISSN 1010-4283, E-ISSN 1423-0380, Vol. 33, no S1, p. 91-91Article in journal (Other academic)
  • 24. Nisman, Benjamin
    et al.
    Allweis, Tanir
    Kadouri, Luna
    Mali, Bela
    Hamburger, Tamar
    Baras, Mario
    Gronowitz, Simon
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Peretz, Tamar
    Comparison of diagnostic and prognostic performance of two assays measuring thymidine kinase 1 activity in serum of breast cancer patients2013In: Clinical Chemistry and Laboratory Medicine, ISSN 1434-6621, E-ISSN 1437-4331, Vol. 51, no 2, p. 439-447Article in journal (Refereed)
    Abstract [en]

    Background: We compared two recently developed immuno assays for serum thymidine kinase 1 (TK1) activity: one manual assay (DiviTum, Biovica (R)) and one fully automated assay (Liaison, Diasorin (R)). Methods: The study included 368 women: 149 healthy blood donors (control), 59 patients with benign breast disease (BBD) and 160 patients with primary breast cancer (BC). Results: A regression analysis of the Liaison (y) and DiviTum (x) assays for all three groups yielded the equation y=3.93+0.03x (r=0.85, n=368). The r-value in BC was higher than in control and BBD (0.90 vs. 0.81 and 0.64). The correlation between the two assays for TK1 values above the cut-off was higher compared to that below (0.88 and 0.59). Breakdown of the BBD group into subgroups with proliferative and non-proliferative lesions was effective only with the measurement of TK1 with DiviTum assay (p=0.03). The TK1 activity determined preoperatively in BC patients with DiviTum and Liaison assays was significantly associated with T-stage (for both p=0.01), presence of vascular invasion (p=0.002 and p=0.02), lack of estrogen receptor (ER) (p=0.001 and p=0.01) and progesterone receptor (PR) (p=0.01 and p=0.03) expression. Only TK1 analyzed with the DiviTum assay was associated with tumor grade and molecular subtype of BC (p=0.02 and p=0.003). Multivariate Cox proportional hazards analyses demonstrated that T-stage, PR status and TK1 activity measured by both methods (DiviTum, RR=3.0, p=0.02 and Liaison, RR=3.1, p=0.01) were independent predictors of disease recurrence. Conclusions: In spite of differences observed between TK1 activity measured by the DiviTum and Liaison assays, both of them may be used for recurrence prediction in preoperative evaluation of BC patients.

  • 25. Nisman, Benjamin
    et al.
    Kadouri, Luna
    Allweis, Tanir
    Maly, Bella
    Hamburger, Tamar
    Gronowitz, Simon
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Peretz, Tamar
    Increased Proliferative Background in Healthy Women with BRCA1/2 Haploinsufficiency Is Associated with High Risk for Breast Cancer2013In: Cancer Epidemiology, Biomarkers and Prevention, ISSN 1055-9965, E-ISSN 1538-7755, Vol. 22, no 11, p. 2110-2115Article in journal (Refereed)
    Abstract [en]

    Previous studies indicated that BRCA haploinsufficiency was associated with activation of the EGF receptor (EGFR) signaling pathway and increased proliferative activity in mammary epithelial cells of healthy women. We hypothesized that these processes might be reflected in the expression of serologic soluble EGFR (sEGFR) and thymidine kinase 1 (TK1) activity, which signal the initial and final steps of the proliferative pathway, respectively. We found that healthy carriers of BRCA1/2 mutations (n = 80) showed a significantly higher TK1 activity than age-matched controls (P = 0.0003), and TK1 activity was similar in women with BRCA1 and BRCA2 mutations (P = 0.74). The sEGFR concentration was significantly higher in women with BRCA1 than in controls and BRCA2 mutation (P = 0.013 and 0.002, respectively). During follow-up, four of 80 BRCA1/2 mutation carriers developed breast cancer. These women showed a significantly higher TK1 activity and somewhat higher sEGFR concentrations than the other 76 BRCA1/2 carriers (P = 0.04 and 0.09, respectively). All tumors were negative for ovarian hormone receptors, but showed a high EGFR expression. This study was limited by the short-term follow-up (mean, 27 months; range, 5-45), which resulted in a small sample size. Women with BRCA1 and BRCA2 mutations that had undergone risk-reducing bilateral salpingo-oophorectomy (BSO) showed significantly lower sEGFR compared with those without surgery (P = 0.007 and 0.038, respectively). Larger, prospective studies are warranted to investigate whether TK1 and sEGFR measurements may be useful for identifying healthy BRCA1/2 carriers with high risk of developing breast cancer; moreover, sEGFR measurements may serve as effective tools for assessing risk before and after BSO.

  • 26. Orozovic, Goran
    et al.
    Järhult, Josef D.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Orozovic, Kanita
    Tolf, Conny
    Latorre-Margalef, Neus
    Lennerstrand, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Olsen, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Oseltamivir- and Zanamivir-Resistance Related Mutations in Influenza A Viruses Isolated from Wild Mallards in Sweden Studied by a Colorimetric Neuraminidase Inhibition AssayManuscript (preprint) (Other academic)
    Abstract [en]

     

    Resistance to neuraminidase inhibitors (NAIs) is a growing problem in the battle against influenza A viruses. However, little is known about the resistance of viruses isolated from dabbling ducks, the natural reservoir of the influenza virus. To date, no low-pathogenic avian influenza (LPAI) virus functionally resistant to NAIs has been detected. The aim of this study was to investigate mallard isolates of influenza A virus previously identified to carry oseltamivir carboxylate (OC)- or zanamivir (ZA)-related resistance mutations. In this work, 21 viruses belonging to the N1, N3, N6 and N9 subtypes were analyzed using a colorimetric NA inhibition assay. The R118K mutation was the most frequently observed; it was seen in all subtypes except for N6. IC50 values confirmed the differences in sensitivity to OC or ZA previously observed in the N1 and N2 groups of NAs. Furthermore, both negative controls (NCs) in the N6 and one NC in the N9 subtype were less sensitive to ZA than were genotypically related mutants of the respective subtypes. The presence of OC- and ZA-related mutations in the NA of viruses isolated from wild birds did not result in a NAI-resistant phenotype in this study. The R118K and R152K mutations seemed to somewhat increase the sensitivity to both NAIs compared to WT viruses which supports the thought that the mutations were a result of natural NA variation and not induced by NAI residuals in the environment. Although not resulting in a NAI resistant phenotype, the finding of the mutations D151N and I222V shows that mutations with the potential to enhance NAI resistance exist in the pool of LPAI viruses which stresses the need for surveillance of NAI resistance in wild birds.

  • 27.
    Palanisamy, Navaneethan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Danielsson, Axel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Kokkula, Chakradhar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Yin, Hong
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Bondeson, Kåre
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Wesslen, Lars
    Duberg, Ann-Sofi
    Lennerstrand, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Implications of baseline polymorphisms for potential resistance to NS3 protease inhibitors in Hepatitis C virus genotypes 1a, 2b and 3a2013In: Antiviral Research, ISSN 0166-3542, E-ISSN 1872-9096, Vol. 99, no 1, p. 12-17Article in journal (Refereed)
    Abstract [en]

    The future interferon-free treatment of hepatitis C virus (HCV) infection could include NS3 protease inhibitors (PIs) for potent pan-genotypic effect. We studied the prevalence of pre-existing PI resistance associated amino acid variants (RAVs) in 126 treatment-naive patient samples of HCV genotypes 1a, 2b and 3a, the most common genotypes in Sweden. The NS3 genes were each amplified by nested PCR method with degenerated primers to enable a broad genotype analysis. Population sequencing method was used, and the sequences were aligned with the NS3 sequence from HCV genotype 1a H77 strain. Interpretation of fold-change resistance to NS3 candidate drugs were done from already published phenotypic resistance data. The prevalence of known PI RAVs at baseline in genotype 1a was 28% (15/53), either single (V36L or Q80K/R) or combinations (T54A/S and V55A/I) of mutation(s). In genotype 2b, specific mutations like V36L, Q80G and S122R of viral NS3 protease gene were found in 100% (11/11). These may be the natural polymorphisms unique to genotype 2b. Similarly, specific mutations like V36L and D168Q were found uniquely in all 3a samples (30/30). The natural PI RAVs found in genotype 1a, although with relatively weak resistance, could still render up to 10-fold-resistance to the approved (boceprevir and telaprevir) and the 2nd generation Pis (faldaprevir and simeprevir). Moreover, the natural polymorphisms in genotype 2b (i.e. S122R) and 3a (i.e. D168Q), with inherent PI drug resistance of up to 20 and 700 fold respectively, would explain why current Pis are primarily directed against genotype 1.

  • 28.
    Palanisamy, Navaneethan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology. Heidelberg Univ, Mol & Cellular Engn Grp, BioQuant, D-69120 Heidelberg, Germany.; Heidelberg Univ, Hartmut Hoffmann Berling Int Grad Sch Mol & Cellu, Heidelberg, Germany.
    Lennerstrand, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Computational prediction of Usutu virus E protein B cell and T cell epitopes for potential vaccine development.2017In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 85, no 5, p. 350-364Article in journal (Refereed)
    Abstract [en]

    Usutu virus (family Flaviviridae), once confined to Africa, has emerged in Europe a decade ago. The virus has been spreading throughout Europe at a greater pace mostly affecting avian species. While most bird species remain asymptomatic carriers of this virus, few bird species are highly susceptible. Lately, Usutu virus (USUV) infections in humans were reported sporadically with severe neuroinvasive symptoms like meningoencephalitis. Since so much is unknown about this virus, which potentially may cause severe diseases in humans, there is a need for more studies of this virus. In the present study, we have used computational tools to predict potential B-cell and T-cell epitopes of USUV envelope (E) protein. We found that amino acids between positions 68 and 84 could be a potential B-cell epitope while amino acids between positions 53 and 69 could be a potential MHC class-I and class-II restricted T-cell epitope. By homology 3D modeling of USUV E protein, we found that the predicted B-cell epitope was predominantly located in the coil region while T-cell epitope was located in the beta strand region of the E protein. Additionally, the potential MHC class-I T-cell epitope (LAEVRSYCYL) was predicted to bind to nearly 24 HLAs (IC50 ≤5000 nM) covering nearly 86.44% of the Black population and 96.90% of the Caucasoid population. Further, in vivo studies are needed to validate the predicted epitopes.

  • 29.
    Pujato, N.
    et al.
    Univ Nacl Litoral, Fac Bioquim & Ciencias Biol, Santa Fe, Argentina.
    Camussone, C. M.
    Consejo Nacl Invest Cient & Tecn, Consejo Nacl Invest Cient & Tecn, Buenos Aires, DF, Argentina;INTA, Estn Expt Agr Rafaela, Santa Fe, Argentina.
    Renna, M. S.
    Consejo Nacl Invest Cient & Tecn, Consejo Nacl Invest Cient & Tecn, Buenos Aires, DF, Argentina;Univ Nacl Litoral, Fac Ciencias Vet, Esperanza, Santa Fe, Argentina.
    Perrig, M. S.
    Univ Nacl Litoral, Fac Bioquim & Ciencias Biol, Santa Fe, Argentina;Consejo Nacl Invest Cient & Tecn, Consejo Nacl Invest Cient & Tecn, Buenos Aires, DF, Argentina.
    Morein, B.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Calvinho, L. F.
    INTA, Estn Expt Agr Rafaela, Santa Fe, Argentina;Univ Nacl Litoral, Fac Ciencias Vet, Esperanza, Santa Fe, Argentina.
    Marcipar, I. S.
    Univ Nacl Litoral, Fac Bioquim & Ciencias Biol, Santa Fe, Argentina;Consejo Nacl Invest Cient & Tecn, Consejo Nacl Invest Cient & Tecn, Buenos Aires, DF, Argentina.
    Evaluation of the humoral immune response to a multicomponent recombinant vaccine against S-aureus in healthy pregnant heifers2018In: The Veterinary Journal, ISSN 1090-0233, E-ISSN 1532-2971, Vol. 235, p. 47-53Article in journal (Refereed)
    Abstract [en]

    Staphylococcus aureus is a worldwide pathogen that causes mastitis in dairy herds. Shortcomings in control programs have encouraged the development of vaccines against this pathogen. This study evaluated the vaccine candidate VacR, which included recombinant S. aureus protein clumping factor A (rClf), fibronectin binding protein A (rFnBP) and hemolysin beta (rBt), formulated with a novel immune-stimulating complex. Comparisons were made between healthy pregnant heifers that received either VacR (n = 8; VacR group) or phosphate buffered saline (PBS) plus adjuvant (control group) SC in the supramammary lymph node area on days 45 and 15 before the expected calving date. Blood and foremilk samples were collected from 7 to 60 days post-calving. After calving, heifers in the VacR group produced higher total IgG (IgG(total)) titers against each component, in both serum (rBt, 3.4 x 10(5); rClf, 3.1 x 10(5); rFnBP, 2.3 x 10(5)) and milk (rBt, 2.6 x 10(4); rClf, 1.3 x 10(4); rFnBP, 1.1 x 10(4)), than control heifers (P < 0.0001). There were increased concentrations of IgG(1), and IgG(2) in VacR group (P < 0.05), in both serum and milk. Humoral responses remained high throughout the period most susceptible to intramammary infections (P < 0.01). Antibodies produced against S. aureus rClf and rFnBP reduced bacterial adherence to fibronectin and fibrinogen by 73% and 67%, respectively (P < 0.001). Milk antibodies against these adhesins inhibited S. aureus invasion of a mammary epithelial cell line (MAC-T), resulting in 15.7% of bacteria internalized (P < 0.0001). There was an approximately 6-fold reduction in the hemolysis titer for the native hemolysin in the VacR group compared to the control group (P < 0.0001) and a significantly increase in the proportion of positive neutrophils (VacR, 29.7%; PBS, 13.1%) and the mean fluorescent index (VacR, 217.4; PBS, 152.6; P < 0.01) in the VacR group. The results suggest that VacR is a valuable vaccine candidate against S. aureus infections, and merits further field trials and experimental challenges. (C) 2018 Elsevier Ltd. All rights reserved.

  • 30. Sauerbrei, A.
    et al.
    Vödisch, S.
    Bohn, K.
    Schacke, M.
    Gronowitz, Simon
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Screening of herpes simplex virus type 1 isolates for acyclovir resistance using DiviTum® assay2013In: Journal of Virological Methods, ISSN 0166-0934, E-ISSN 1879-0984, Vol. 188, no 1-2, p. 70-72Article in journal (Refereed)
    Abstract [en]

    Rapid alternative methods are required to evaluate easily acyclovir (ACV) sensitivity of clinical herpes simplex virus (HSV) isolates. The objective of this study was to screen 54 ACV-sensitive and 41 ACV-resistant clinical HSV-1 isolates, well characterized by phenotypic and genotypic methods, for the phosphorylation activity of the viral thymidine kinase (TK) using a commercially available and modified non-radioactive DiviTum® test on the basis of an indirect enzyme linked immunosorbent assay. The ACV-sensitive HSV-1 isolates had high TK activity values between 31.5±6.4 DiviTum® Units per liter (DU/L) and 487.4±60.1DU/L. The mean activity of all ACV-sensitive isolates was calculated as 212.3±15.7 DU/L. By contrast, the mean activity of all ACV-resistant HSV-1 isolates was significantly lower at 5.5±1.3DU/L. Out of the 41 ACV-resistant HSV-1 isolates, 38 had no or very low phosphorylation activities of the viral TK between 0DU/L and 9.3±3.2DU/L. The remaining three ACV-resistant viral isolates had TK activities between 44.6±5.1DU/L and 80.9±13.3DU/L. In conclusion, the modified DiviTum® test can be used to screen HSV-1 isolates for their sensitivity to ACV. Acyclovir-sensitive HSV-1 isolates show TK activities &gt;30DU/L and ACV-resistant isolates have activity values &lt;10DU/L. However, single ACV-resistant HSV-1 isolates can have TK activity values &gt;30DU/L. These strains are most likely ACV-resistant TK-altered mutants, but no evidence was provided for an alteration of the TK.

  • 31.
    Stalhandske, Per
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Wang, Liya
    Westberg, Sara
    von Euler, Henrik
    Groth, Erika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Gustafsson, Sven A.
    Eriksson, Staffan
    Lennerstrand, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine, Clinical Virology.
    Homogeneous assay for real-time and simultaneous detection of thymidine kinase 1 and deoxycytidine kinase activities2013In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 432, no 2, p. 155-164Article in journal (Refereed)
    Abstract [en]

    Measurement of thymidine kinase-1 (TK1) and deoxycytidine kinase (dCK) activity may be useful in cancer disease management. Therefore, a one-step homogeneous assay for real-time determination of TK1 and dCK was developed by combining enzyme complementation with fluorescent signal generation using primer extension and a quenched probe oligodeoxyribonucleotide system at 37 degrees C. Complementation, for producing dCTP and TIP from nucleoside substrates, was carried out by dTMP kinase and/or UMP/CMP kinase and nucleoside diphosphate kinase. dNTP was continuously incorporated into a fixed oligodeoxyribonucleotide primer, template, and probe system, and the fluorescent signal was generated by using the combined actions of primer extension and 5' exonuclease activity of Thermophilus aquaticus (Taq) DNA polymerase for specific relief of fluorescent quenching. Fluorescence was captured at 1-min intervals using a real-time polymerase chain reaction (PCR) instrument. A horizontal threshold line, crossing all sample relative fluorescent units (RFU) values at the level of the RFU of the blank sample at the end of the assay (i.e., 90 min), was drawn, obtaining RFU measurement data in minutes for each sample. Duplex proof of principle was demonstrated by the independent determination of different amounts of dCK and TK1 in combination. R-2 values of 0.90 were demonstrated with Prolifigen TK-REA U/L reference values obtained from pathological canine and human serum samples.

  • 32. 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)
  • 33.
    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.

  • 34.
    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.

  • 35.
    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.

1 - 35 of 35
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