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Sample records for b19 genotype specific

  1. Phylogenetic analysis of human parvovirus B19, indicating two subgroups of genotype 1 in Vietnamese patients.

    PubMed

    Toan, Nguyen L; Duechting, Anja; Kremsner, Peter G; Song, Le H; Ebinger, Martin; Aberle, Susanne; Binh, Vu Q; Duy, Dinh Ng; Torresi, Joseph; Kandolf, Reinhard; Bock, C-Thomas

    2006-10-01

    Recently, three distinct genotypes (1, 2 and 3) of human parvovirus B19 (B19) have been identified. However, the characteristics and distribution of B19 genotypes in Vietnam have not been investigated. Phylogenetic analysis using 49 subgenomic NS1/VP1u regions and two coding NS1-VP1/VP2 regions has been applied to investigate the prevalence of B19 genotypes in Vietnamese patients co-infected with Hepatitis B virus. Genetic analysis of the subgenomic NS1/VP1u region of B19 revealed that two genotypes of B19 were identified in these populations, with predominance of genotype 1 (47/49, 96 %) followed by genotype 2 (2/49, 4 %), but not genotype 3. Further, phylogenetic analysis of subgenomic B19 genomes revealed two major subgroups within genotype 1 (B19-1A and B19-1B) with an estimated nucleotide difference of >5 % between each subgroup, forming different branches. The mean percentage of amino acid variation between subgroup B19-1A and B19-1B was >2 % of the NS1, VP1 and VP2 proteins. Our results indicated that two of the three known genotypes of B19 were present in Vietnamese patients, with genotype 1 predominating, and that this genotype can be classified into at least two subgroups, B19-1A and B19-1B.

  2. Identification and characterization of acute infection with parvovirus B19 genotype 2 in immunocompromised patients in Poland.

    PubMed

    Grabarczyk, Piotr; Kalińska, Aleksandra; Kara, Mahmut; Wieczorek, Renata; Ejduk, Anna; Sulkowska, Ewa; Gołębiowska-Staroszczyk, Sydonia; Matysiak, Michał; Baylis, Sally A; Brojer, Ewa

    2011-01-01

    Parvovirus B19 (B19V) is divided into three genotypes. Genotypes 2 and 3 may cause diagnostic difficulties and their epidemiology is not well understood. In the present study the prevalence of B19V genotypes in patients with symptomatic infection in Poland was evaluated and the course of infection in patients infected with non-genotype 1 strains is described. Real-time PCR, able to detect all three genotypes of B19V was used to screen patient plasma samples. Sixty-nine, mainly acute-phase B19V DNA positive cases were identified in patients from hematological and obstetric/gynecological wards between 2004 and 2008. Thirty patients were studied in greater detail and genotyping was performed by analysis of the NS1/VP1u region. The majority of samples were genotype 1. However two (6.6%) strains were identified as genotype 2, associated with high viremia and identified in a kidney transplant recipient with anemia and a leukemia patient, following chemotherapy, with pancytopenia. A change of immunosuppression treatment in the former and treatment with intravenous immunoglobulin in latter, resulted in normalization of clinical parameters, and whilst viral loads fell, B19V DNA was still detectable. The kidney transplant recipient subsequently became pregnant with no clinical complications, although persistently infected with B19V genotype 2. This is the first description of symptomatic cases of genotype 2 B19V infection in Eastern Europe suggesting that acute infection, particularly among immunocompromised patients with these virus strains may be more prevalent than thought.

  3. Insights into epidemiology of human parvovirus B19 and detection of an unusual genotype 2 variant, Bulgaria, 2004 to 2013.

    PubMed

    Ivanova, Stefka Krumova; Mihneva, Zafira Georgieva; Toshev, Andon Krumov; Kovaleva, Valentina Pavlova; Andonova, Lubena Georgieva; Muller, Claude P; Hübschen, Judith M

    2016-01-01

    The present study aimed to determine the role of human parvovirus В19 (B19V) as an aetiological agent in measles and rubella negative fever/rash patients from Bulgaria between 2004 and 2013. A total of 1,266 sera from all over the country were tested for B19V IgM antibodies and all positives were further investigated by polymerase chain reaction (PCR). Overall, 280 sera (22%) were B19V IgM positive and 227 of these (81%) were also PCR positive. The highest number of IgM positives was found among five to nine year-old children (27%). Eight infected women gave birth to healthy children; one fetus was aborted with hydrops fetalis. Of the 55 genetic sequences obtained, 54 belonged to genotype 1a and one grouped as a genotype 2 outlier. Phylogenetic analysis of all available genotype 2 sequences covering the 994 nucleotide non-structural protein 1(NS1)/capsid viral protein 1 (VP1) unique region junction, showed that only one other sequence grouped with the outlier strain, forming a clearly distinct and well-supported cluster of genotype 2 (between-group genetic distance: 3.32%). In accordance with B19V nomenclature, this cluster may represent a new subgenotype 2b. The study showed that B19V infections may be falsely identified as rubella or measles in ca 22% of cases, emphasising the need for laboratory confirmation. PMID:26847955

  4. Granzyme B mediated function of Parvovirus B19-specific CD4+ T cells

    PubMed Central

    Kumar, Arun; Perdomo, Maria F; Kantele, Anu; Hedman, Lea; Hedman, Klaus; Franssila, Rauli

    2015-01-01

    A novel conception of CD4+ T cells with cytolytic potential (CD4+ CTL) is emerging. These cells appear to have a part in controlling malignancies and chronic infections. Human parvovirus B19 can cause a persistent infection, yet no data exist on the presence of B19-specific CD4+ CTLs. Such cells could have a role in the pathogenesis of some autoimmune disorders reported to be associated with B19. We explored the cytolytic potential of human parvovirus B19-specific T cells by stimulating peripheral blood mononuclear cell (PBMC) with recombinant B19-VP2 virus-like particles. The cytolytic potential was determined by enzyme immunoassay-based quantitation of granzyme B (GrB) and perforin from the tissue culture supernatants, by intracellular cytokine staining (ICS) and by detecting direct cytotoxicity. GrB and perforin responses with the B19 antigen were readily detectable in B19-seropositive individuals. T-cell depletion, HLA blocking and ICS experiments showed GrB and perforin to be secreted by CD4+ T cells. CD4+ T cells with strong GrB responses were found to exhibit direct cytotoxicity. As anticipated, ICS of B19-specific CD4+ T cells showed expected co-expression of GrB, perforin and interferon gamma (IFN-γ). Unexpectedly, also a strong co-expression of GrB and interleukin 17 (IL-17) was detected. These cells expressed natural killer (NK) cell surface marker CD56, together with the CD4 surface marker. To our knowledge, this is the first report on virus-specific CD4+ CTLs co-expressing CD56 antigen. Our results suggest a role for CD4+ CTL in B19 immunity. Such cells could function within both immune regulation and triggering of autoimmune phenomena such as systemic lupus erythematosus (SLE) or rheumatoid arthritis. PMID:26246896

  5. Prevalence of parvovirus B19 specific antibody in pregnant women with spontaneous abortion.

    PubMed

    Rahbar, Nahid; Vali Zadeh, Saeid; Ghorbani, Raheb; Kheradmand, Pegah

    2015-01-01

    Human parvovirus B19 is a very common viral infection especially in school-aged children. The infection during pregnancy can affect the fetus due to lack of mother's immunity. Although, there is still no evidence of fetal teratogenic effects with parvovirus B19, but non-immune fetal hydrops and abortion may be caused by vertical transmission of the virus during pregnancy. This study was aimed to assess the prevalence of parvovirus B19-specific antibody (IgM) in pregnant women who had a spontaneous abortion. This cross-sectional study was carried out in all pregnant women who referred due to a spontaneous abortion. All demographic information such as age, occupation, and gestational age, last history of abortion, gravity, and presence of children below the age of six was recorded and a blood sample was provided for all the women. Then, the blood samples were tested to assay parvovirus B19-specific antibody (IgM) by EuroImmune ELISA kit. Among 94 pregnant women with the mean age of 28.4 years who had a spontaneous abortion, parvovirus B19 specific antibody (IgM) was detected in 17 participants (18.1%). Meanwhile, 14 women (14.9%) were suspected for presence of the antibody in their blood sample. There was no significant difference between the presence of antibody and age of pregnant women, occupation, gestational age, number of previous abortion, presence of children below the age of six and number of pregnancy. These findings revealed that a high percentage of pregnant women are probably non-immune against parvovirus B19, and also there might be a number of spontaneous abortions in which parvovirus infection caused fetal death.  However, more studies are needed to prove the absolute role of parvovirus B19 in these abortions.

  6. Genotyping of human parvovirus B19 in clinical samples from Brazil and Paraguay using heteroduplex mobility assay, single-stranded conformation polymorphism and nucleotide sequencing.

    PubMed

    Mendonça, Marcos César Lima de; Ferreira, Ana Maria de Amorim; Santos, Marta Gonçalves Matos dos; Oviedo, Elva Cristina; Bello, Maria Sônia Dal; Siqueira, Marilda Mendonça; Maceira, Juan Manuel Piñeiro; von Hubinger, Maria Genoveva; Couceiro, José Nelson dos Santos Silva

    2011-06-01

    Heteroduplex mobility assay, single-stranded conformation polymorphism and nucleotide sequencing were utilised to genotype human parvovirus B19 samples from Brazil and Paraguay. Ninety-seven serum samples were collected from individuals presenting with abortion or erythema infectiosum, arthropathies, severe anaemia and transient aplastic crisis; two additional skin samples were collected by biopsy. After the procedure, all clinical samples were classified as genotype 1.

  7. Specific Targeting of Proerythroblasts and Erythroleukemic Cells by the VP1u Region of Parvovirus B19.

    PubMed

    Leisi, Remo; von Nordheim, Marcus; Kempf, Christoph; Ros, Carlos

    2015-09-16

    Viruses are evolutionarily developed cell-entering nanomachines, which are frequently used as gene or drug delivery systems. Parvovirus B19 (B19V) shows a remarkably restricted tropism for erythropoietin-dependent erythroid differentiation stages, and thus this virus provides an opportunity to deliver cargo to these intermediate differentiated cells. Here we report the construction of a delivery system from B19V subunits that maintains the highly selective cell-entry of the native virus and offers versatile cargo transport. To obtain this specific carrier, we conjugated the cell-targeting VP1u region of B19V to NeutrAvidin as a loading platform for biotinylated cargos. The VP1u-NeutrAvidin conjugate delivered fluorophores, DNA, and toxic payloads specifically to erythroid cells around the proerythroblast differentiation stage, including erythroleukemic cells. The VP1u-NeutrAvidin represents a unique cell surface marker which exclusively detects intermediate erythroid differentiation stages. Furthermore, the cell-entering property of this viral-based targeting system offers opportunities for erythroid-specific drug delivery or gene therapy.

  8. Generation of multifunctional murine monoclonal antibodies specifically directed to the VP1unique region protein of human parvovirus B19.

    PubMed

    Drechsler, Maik D; Obermeier, Ingrid; Döring, Yvonne; Lackner, Karl J; Modrow, Susanne; von Landenberg, Philipp

    2008-01-01

    Little is known about the VP1unique region (VP1u), a part of one major capsid protein of human parvovirus B19 (B19), concerning its involvement in viral replication and infection cycle. Showing a phospholipase A2 (PLA2)-like activity, which is discussed to be necessary for viral release from host cell, its precise function remains unclear. The purpose of this study was to generate multifunctional monoclonal antibodies (mabs) for different applications that may be useful in investigating VP1u's relevance. To establish antiVP1u antibodies, spleen cells from Balb/c mice immunized with purified recombinant viral protein were used for generating antibody-producing hybridoma cell lines. Usability of the antibodies was tested in enzyme-linked immunosorbent assay (ELISA), Western-blot analysis, immunofluorescence and an inhibition assay of enzymatic activity of PLA2. Three hybridoma cell lines secreting mab's specifically directed against the VP1u protein of B19 could be generated and functioned in every screening method used in this study. These antibodies are helpful tools for investigations in B19 research and diagnosis. Furthermore, the antibodies could help in gaining a deeper understanding of VP1u's role in viral replication and infection especially in the importance of its constitutive PLA2-like activity.

  9. Molecular phenotypes of human parvovirus B19 in patients with myocarditis

    PubMed Central

    Bock, C-Thomas; Düchting, Anja; Utta, Friederike; Brunner, Eva; Sy, Bui Tien; Klingel, Karin; Lang, Florian; Gawaz, Meinrad; Felix, Stephan B; Kandolf, Reinhard

    2014-01-01

    AIM: To investigate molecular phenotypes of myocardial B19V-infection to determine the role of B19V in myocarditis and dilated cardiomyopathy (DCM). METHODS: Endomyocardial biopsies (EMBs) from 498 B19V-positive patients with myocarditis and DCM were analyzed using molecular methods and functional experiments. EMBs were obtained from the University Hospitals of Greifswald and Tuebingen and additionally from 36 German cardiology centers. Control tissues were obtained at autopsy from 34 victims of accidents, crime or suicide. Identification of mononuclear cell infiltrates in EMBs was performed using immunohistological staining. Anti-B19V-IgM and anti-B19V-IgG were analyzed by enzyme-linked immunosorbent assay (ELISA). B19V viral loads were determined using in-house quantitative real-time polymerase chain reaction (PCR). For B19V-genotyping a new B19V-genotype-specific restriction fragment length polymorphism (RFLP)-PCR was established. B19V-genotyping was verified by direct DNA-sequencing and sequences were aligned using BLAST and BioEdit software. B19V P6-promoter and HHV6-U94-transactivator constructs were generated for cell culture experiments. Transfection experiments were conducted using human endothelial cells 1. Luciferase reporter assays were performed to determine B19V-replication activity. Statistical analysis and graphical representation were calculated using SPSS and Prism5 software. RESULTS: The prevalence of B19V was significantly more likely to be associated with inflammatory cardiomyopathy (iCMP) compared to uninflamed DCM (59.6% vs 35.3%) (P < 0.0001). The detection of B19V-mRNA replication intermediates proved that replication of B19V was present. RFLP-PCR assays showed that B19V-genotype 1 (57.4%) and B19V-genotype 2 (36.7%) were the most prevalent viral genotypes. B19V-genotype 2 was observed more frequently in EMBs with iCMP (65.0%) compared to DCM (35%) (P = 0.049). Although there was no significant difference in gender-specific B19V

  10. Genotype Specification Language.

    PubMed

    Wilson, Erin H; Sagawa, Shiori; Weis, James W; Schubert, Max G; Bissell, Michael; Hawthorne, Brian; Reeves, Christopher D; Dean, Jed; Platt, Darren

    2016-06-17

    We describe here the Genotype Specification Language (GSL), a language that facilitates the rapid design of large and complex DNA constructs used to engineer genomes. The GSL compiler implements a high-level language based on traditional genetic notation, as well as a set of low-level DNA manipulation primitives. The language allows facile incorporation of parts from a library of cloned DNA constructs and from the "natural" library of parts in fully sequenced and annotated genomes. GSL was designed to engage genetic engineers in their native language while providing a framework for higher level abstract tooling. To this end we define four language levels, Level 0 (literal DNA sequence) through Level 3, with increasing abstraction of part selection and construction paths. GSL targets an intermediate language based on DNA slices that translates efficiently into a wide range of final output formats, such as FASTA and GenBank, and includes formats that specify instructions and materials such as oligonucleotide primers to allow the physical construction of the GSL designs by individual strain engineers or an automated DNA assembly core facility. PMID:26886161

  11. Genotype Specification Language.

    PubMed

    Wilson, Erin H; Sagawa, Shiori; Weis, James W; Schubert, Max G; Bissell, Michael; Hawthorne, Brian; Reeves, Christopher D; Dean, Jed; Platt, Darren

    2016-06-17

    We describe here the Genotype Specification Language (GSL), a language that facilitates the rapid design of large and complex DNA constructs used to engineer genomes. The GSL compiler implements a high-level language based on traditional genetic notation, as well as a set of low-level DNA manipulation primitives. The language allows facile incorporation of parts from a library of cloned DNA constructs and from the "natural" library of parts in fully sequenced and annotated genomes. GSL was designed to engage genetic engineers in their native language while providing a framework for higher level abstract tooling. To this end we define four language levels, Level 0 (literal DNA sequence) through Level 3, with increasing abstraction of part selection and construction paths. GSL targets an intermediate language based on DNA slices that translates efficiently into a wide range of final output formats, such as FASTA and GenBank, and includes formats that specify instructions and materials such as oligonucleotide primers to allow the physical construction of the GSL designs by individual strain engineers or an automated DNA assembly core facility.

  12. T helper cell-mediated interferon-gamma expression after human parvovirus B19 infection: persisting VP2-specific and transient VP1u-specific activity.

    PubMed

    Franssila, R; Auramo, J; Modrow, S; Möbs, M; Oker-Blom, C; Käpylä, P; Söderlund-Venermo, M; Hedman, K

    2005-10-01

    Human parvovirus B19 is a small non-enveloped DNA virus with an icosahedral capsid consisting of proteins of only two species, the major protein VP2 and the minor protein VP1. VP2 is contained within VP1, which has an additional unique portion (VP1u) of 227 amino acids. We determined the ability of eukaryotically expressed parvovirus B19 virus-like particles consisting of VP1 and VP2 in the ratio recommended for vaccine use, or of VP2 alone, to stimulate, in an HLA class II restricted manner, peripheral blood mononuclear cells (PBMC) to proliferate and to secrete interferon gamma (IFN-gamma) and interleukin (IL)-10 cytokines among recently and remotely B19 infected subjects. PBMC reactivity with VP1u was determined specifically with a prokaryotically expressed VP1u antigen. In general, B19-specific IFN-gamma responses were stronger than IL-10 responses in both recent and remote infection; however, IL-10 responses were readily detectable among both groups, with the exception of patients with relapsed or persisting symptoms who showed strikingly low IL-10 responses. Whereas VP1u-specific IFN-gamma responses were very strong among the recently infected subjects, the VP1u-specific IFN-gamma and IL-10 responses were virtually absent among the remotely infected subjects. The disappearance of VP1u-specific IFN-gamma expression is surprising, as B-cell immunity against VP1u is well maintained.

  13. T helper cell-mediated interferon-gamma expression after human parvovirus B19 infection: persisting VP2-specific and transient VP1u-specific activity

    PubMed Central

    Franssila, R; Auramo, J; Modrow, S; Möbs, M; Oker-Blom, C; Käpylä, P; Söderlund-Venermo, M; Hedman, K

    2005-01-01

    Human parvovirus B19 is a small non-enveloped DNA virus with an icosahedral capsid consisting of proteins of only two species, the major protein VP2 and the minor protein VP1. VP2 is contained within VP1, which has an additional unique portion (VP1u) of 227 amino acids. We determined the ability of eukaryotically expressed parvovirus B19 virus-like particles consisting of VP1 and VP2 in the ratio recommended for vaccine use, or of VP2 alone, to stimulate, in an HLA class II restricted manner, peripheral blood mononuclear cells (PBMC) to proliferate and to secrete interferon gamma (IFN-γ) and interleukin (IL)-10 cytokines among recently and remotely B19 infected subjects. PBMC reactivity with VP1u was determined specifically with a prokaryotically expressed VP1u antigen. In general, B19-specific IFN-γ responses were stronger than IL-10 responses in both recent and remote infection; however, IL-10 responses were readily detectable among both groups, with the exception of patients with relapsed or persisting symptoms who showed strikingly low IL-10 responses. Whereas VP1u-specific IFN-γ responses were very strong among the recently infected subjects, the VP1u-specific IFN-γ and IL-10 responses were virtually absent among the remotely infected subjects. The disappearance of VP1u-specific IFN-γ expression is surprising, as B-cell immunity against VP1u is well maintained. PMID:16178856

  14. Tracking of Peptide-Specific CD4+ T-Cell Responses after an Acute Resolving Viral Infection: a Study of Parvovirus B19

    PubMed Central

    Kasprowicz, Victoria; Isa, Adiba; Tolfvenstam, Thomas; Jeffery, Katie; Bowness, Paul; Klenerman, Paul

    2006-01-01

    The evolution of peptide-specific CD4+ T-cell responses to acute viral infections of humans is poorly understood. We analyzed the response to parvovirus B19 (B19), a ubiquitous and clinically significant pathogen with a compact and conserved genome. The magnitude and breadth of the CD4+ T-cell response to the two B19 capsid proteins were investigated using a set of overlapping peptides and gamma interferon-specific enzyme-linked immunospot assays of peripheral blood mononuclear cells (PBMCs) from a cohort of acutely infected individuals who presented with acute arthropathy. These were compared to those for a cohort of B19-specific immunoglobulin M-negative (IgM−), IgG+ remotely infected individuals. Both cohorts of individuals were found to make broad CD4+ responses. However, while the responses following acute infection were detectable ex vivo, responses in remotely infected individuals were only detected after culture. One epitope (LASEESAFYVLEHSSFQLLG) was consistently targeted by both acutely (10/12) and remotely (6/7) infected individuals. This epitope was DRB1*1501 restricted, and a major histocompatibility complex peptide tetramer stained PBMCs from acutely infected individuals in the range of 0.003 to 0.042% of CD4+ T cells. Tetramer-positive populations were initially CD62Llo; unlike the case for B19-specific CD8+ T-cell responses, however, CD62L was reexpressed at later times, as responses remained stable or declined slowly. This first identification of B19 CD4+ T-cell epitopes, including a key immunodominant peptide, provides the tools to investigate the breadth, frequency, and functions of cellular responses to this virus in a range of specific clinical settings and gives an important reference point for analysis of peptide-specific CD4+ T cells during acute and persistent virus infections of humans. PMID:16943301

  15. Parvovirus B19 Test

    MedlinePlus

    ... Viral detection involves finding parvovirus B19 genetic material ( DNA ) in a blood sample or, less commonly, in ... fetal cord blood, or amniotic fluid . Parvovirus B19 DNA testing is performed primarily to detect active parvovirus ...

  16. Advances in Human B19 Erythrovirus Biology▿

    PubMed Central

    Servant-Delmas, Annabelle; Lefrère, Jean-Jacques; Morinet, Frédéric; Pillet, Sylvie

    2010-01-01

    Since its discovery, human parvovirus B19 (B19V), now termed erythrovirus, has been associated with many clinical situations (neurological and myocardium infections, persistent B19V DNAemia) in addition to the prototype clinical manifestations, i.e., erythema infectiosum and erythroblastopenia crisis. In 2002, the use of new molecular tools led to the characterization of three different genotypes of human B19 erythrovirus. Although the genomic organization is conserved, the geographic distribution of the different genotypes varies worldwide, and the nucleotidic divergences can impact the molecular diagnosis of B19 virus infection. The cell cycle of the virus remains partially unresolved; however, recent studies have shed light on the mechanism of cell entry and the interactions of B19V proteins with apoptosis pathways. PMID:20631151

  17. Bone marrow necrosis and fat embolism syndrome in sickle cell disease: increased susceptibility of patients with non-SS genotypes and a possible association with human parvovirus B19 infection.

    PubMed

    Tsitsikas, Dimitris A; Gallinella, Giorgio; Patel, Sneha; Seligman, Henry; Greaves, Paul; Amos, Roger J

    2014-01-01

    Fat embolism syndrome (FES) due to extensive bone marrow necrosis (BMN) in sickle cell disease (SCD) is a potentially under-diagnosed complication associated with severe morbidity and mortality. We identified 58 cases reported in the world literature to date. Typically, patients presented with a seemingly uncomplicated vaso-occlusive crisis (VOC) and subsequently deteriorated rapidly with a drop in their haemoglobin and platelets, development of respiratory failure, encephalopathy and varying degrees of involvement of other systems. Overall mortality in the reported cases was 64% but differed according to the use of transfusion and was 29%, 61% and 91% for patients receiving exchange, top-up or no transfusion respectively. Patients most at risk appear to be those with a "milder" form of SCD as 81% of patients had a genotype other than HbSS and the majority had no history of significant sickle-related complications. Human parvovirus B19 (HPV B19) infection was documented in 24% of cases.

  18. Estimating Genotype- and Environment-Specific Heritabilities

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The advantages of computing genotype- and environment-specific heritabilities are discussed. A statistical approach is used in which logvariances of both genotype by environment interaction and error are modeled as random variables. Resulting estimators of variances are weighted averages of a pool...

  19. Parvovirus B19 1A complete genome from a fatal case in Brazil

    PubMed Central

    Conteville, Liliane Costa; Zanella, Louise; Marín, Michel Abanto; de Filippis, Ana Maria Bispo; Nogueira, Rita Maria Ribeiro; Vicente, Ana Carolina Paulo; de Mendonça, Marcos César Lima

    2015-01-01

    Parvovirus B19 (B19V) infects individuals worldwide and is associated with an ample range of pathologies and clinical manifestations. B19V is classified into three distinct genotypes, all identified in Brazil. Here, we report a complete sequence of a B19V genotype 1A that was obtained by high-throughput metagenomic sequencing. This genome provides information that will contribute to the studies on B19V epidemiology and evolution. PMID:26517666

  20. Diagnostic and prognostic value of molecular and serological investigation of human parvovirus B19 infection during pregnancy.

    PubMed

    Zavattoni, Maurizio; Paolucci, Stefano; Sarasini, Antonella; Tassis, Beatrice; Rustico, Mariangela; Quarenghi, Aida; Piralla, Antonio; Baldanti, Fausto

    2016-09-01

    To define diagnostic and prognostic markers of parvovirus B19 (B19V) fetal infection, two groups were investigated: 1) pregnant women with specific symptoms or contacts with symptomatic households (n=37); 2) mothers with pathological ultrasound findings and the relevant fetus at the time of prenatal diagnosis (n=16). In the first group, diagnosis of B19V infection was achieved using IgM detection in 29/37 (78.3%) of patients, while B19V DNA was detected in 36/37 (97.3%) of infected women. In the second group, intrauterine infection was investigated by amniocentesis (n=5), cordocentesis (n=3) or both (n=5). Median B19V DNA load in amniotic fluid was 8.2x107 copies/ml and in fetal blood was 2x109 copies/ml. Maternal blood was positive for B19V DNA (median 3.8x104 copies/ml) in 14/16 (87.5%) women examined. At time of fetal US investigation, all mothers were B19V IgG positive and B19V IgM were detected in 10/16 (62.5%), while fetal B19V IgG and IgM were detected in 1/8 (12.5%) and 5/8 (62.5%), respectively. Phylogenetic analysis revealed that all B19V maternal and fetal strains belonged to genotype 1A. Diagnosis of maternal, fetal and neonatal B19V infection should be based on both IgM and DNA detection. Prognostic markers of congenital B19V infection need to be defined. PMID:27602415

  1. Update of the human parvovirus B19 biology.

    PubMed

    Servant-Delmas, A; Morinet, F

    2016-02-01

    Since its discovery, the human parvovirus B19 (B19V) has been associated with many clinical situations in addition to the prototype clinical manifestations, i.e. erythema infectiosum and erythroblastopenia crisis. The clinical significance of the viral B19V DNA persistence in sera after acute infection remains largely unknown. Such data may constitute a new clinical entity and is discussed in this manuscript. In 2002, despite the genetic diversity among B19V viruses has been reported to be very low, the description of markedly distinct sequences showed a new organization into three genotypes. The most recent common ancestor for B19V genotypes was estimated at early 1800s. B19V replication is enhanced by hypoxia and this might to explain the high viral load detected by quantitative PCR in the sera of infected patients. The minimum infectious dose necessary to transmit B19V infection by the transfusion of labile blood products remains unclear. At the opposite, the US Food and Drug Administration proposed a limit of 10(4)IU/mL of viral DNA in plasma pools used for the production of plasma derivatives. Recently, a new human parvovirus (PARV4) has been discovered. The consequences on blood transfusion of this blood-borne agent and its pathogenicity are still unknown. PMID:26778837

  2. 7 CFR 15b.19 - New construction.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Uniform Federal Accessibility Standards (USAF) (appendix A to 41 CFR subpart 101-19.6) shall be deemed to... 7 Agriculture 1 2010-01-01 2010-01-01 false New construction. 15b.19 Section 15b.19 Agriculture... ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Accessibility § 15b.19 New construction. (a) Design...

  3. 7 CFR 15b.19 - New construction.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Uniform Federal Accessibility Standards (USAF) (appendix A to 41 CFR subpart 101-19.6) shall be deemed to... 7 Agriculture 1 2011-01-01 2011-01-01 false New construction. 15b.19 Section 15b.19 Agriculture... ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Accessibility § 15b.19 New construction. (a) Design...

  4. 18 CFR 1b.19 - Submissions.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Submissions. 1b.19 Section 1b.19 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.19 Submissions. In the event...

  5. 18 CFR 1b.19 - Submissions.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Submissions. 1b.19 Section 1b.19 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.19 Submissions. In the event...

  6. 18 CFR 1b.19 - Submissions.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 18 Conservation of Power and Water Resources 1 2014-04-01 2014-04-01 false Submissions. 1b.19 Section 1b.19 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.19 Submissions. In the event...

  7. 18 CFR 1b.19 - Submissions.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 18 Conservation of Power and Water Resources 1 2012-04-01 2012-04-01 false Submissions. 1b.19 Section 1b.19 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.19 Submissions. In the event...

  8. 18 CFR 1b.19 - Submissions.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 18 Conservation of Power and Water Resources 1 2011-04-01 2011-04-01 false Submissions. 1b.19 Section 1b.19 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.19 Submissions. In the event...

  9. 7 CFR 15b.19 - New construction.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Uniform Federal Accessibility Standards (USAF) (appendix A to 41 CFR subpart 101-19.6) shall be deemed to... 7 Agriculture 1 2013-01-01 2013-01-01 false New construction. 15b.19 Section 15b.19 Agriculture... ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Accessibility § 15b.19 New construction. (a) Design...

  10. 7 CFR 15b.19 - New construction.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Uniform Federal Accessibility Standards (USAF) (appendix A to 41 CFR subpart 101-19.6) shall be deemed to... 7 Agriculture 1 2014-01-01 2014-01-01 false New construction. 15b.19 Section 15b.19 Agriculture... ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Accessibility § 15b.19 New construction. (a) Design...

  11. Lymphoproliferative responses after infection with human parvovirus B19.

    PubMed Central

    von Poblotzki, A; Gerdes, C; Reischl, U; Wolf, H; Modrow, S

    1996-01-01

    Immunity after infection with the parvovirus B19 is assumed to be conferred by a humoral immune response with development of neutralizing antibody. In contrast, little is known about the nature of T-cell-mediated responses to parvovirus B19 infection in humans. We used recombinant proteins VP1, VP2, and NS1, as well as a recombinant VP1-specific amino-terminal sequence, to test the proliferative responses of peripheral blood mononuclear cells after infection of otherwise healthy individuals with parvovirus B19. These proteins were used as antigens for the stimulation of freshly isolated cells. The results show that a B19 virus-specific cellular immunity develops that is directed against the capsid proteins VP1 and VP2. We also demonstrate that viral determinants are presented to CD4+ T cells by HLA class II molecules. PMID:8794392

  12. Comparison of Three Commercially Available Serologic Assays Used To Detect Human Parvovirus B19-Specific Immunoglobulin M (IgM) and IgG Antibodies in Sera of Pregnant Women

    PubMed Central

    Butchko, Allyson R.; Jordan, Jeanne A.

    2004-01-01

    A split-sample study was conducted to evaluate the performances of three enzyme immunoassays (EIAs) utilizing one or more conformational antigens to detect human parvovirus B19 (B19V)-specific immunoglobulin M (IgM) or IgG in the sera of 198 pregnant women. We compared EIAs available from Biotrin International, Inc. (Dublin, Ireland), Medac Diagnostika (Wedel, Germany), and Mikrogen (Martinsried, Germany). Specimens with discordant results were analyzed further using an immunofluorescence assay (Biotrin). Equivocal data accounted for close to half of all the discrepant results for both IgM and IgG, with 7 of 15 discrepant results from the Medac and Mikrogen kits involving equivocal data and the Biotrin kit giving a single equivocal result. For each specimen, a consensus was established from the four test results if agreement occurred among at least three of four results. Overall, the highest percentage of agreement with the consensus results was seen when Biotrin kits were used; 194 (100%) of 194 and 194 (99.5%) of 195 results for IgM and IgG, respectively, agreed with the consensus results. When Medac kits were used, 189 (97.4%) of 194 and 191 (97.9%) of 195 results for IgM and IgG, respectively, agreed with the consensus, and when Mikrogen kits were used, 179 (92.3%) of 194 and 193 (99%) of 195 results for IgM and IgG, respectively, agreed with the consensus. Given the consensus results, the Medac EIA appeared to generate presumed false-positive results for IgM and the Mikrogen EIA appeared to generate presumed false-positive results for IgG and IgM. In summary, the Biotrin EIAs produced far fewer equivocal results than the other assays and results of the Biotrin EIAs agreed more often with the consensus results than did those of the other commercially available EIAs for detecting B19V-specific IgM and IgG antibodies. PMID:15243081

  13. Global Co-Existence of Two Evolutionary Lineages of Parvovirus B19 1a, Different in Genome-Wide Synonymous Positions

    PubMed Central

    van Binnendijk, Rob S.; Boot, Hein J.; Zaaijer, Hans L.

    2012-01-01

    Parvovirus B19 (B19V) can cause infection in humans. To date, three genotypes of B19V, with subtypes, are known, of which genotype 1a is the most prevalent genotype in the Western world. We sequenced the genome of B19V strains of 65 asymptomatic, recently infected Dutch blood donors, to investigate the spatio-temporal distribution of B19V strains, in the years 2003–2009. The sequences were compared to B19V sequences from Dutch patients with fifth disease, and to global B19V sequences as available from GenBank. All Dutch B19V strains belonged to genotype 1a. Phylogenetic analysis of the strains from Dutch blood donors showed that two groups of genotype 1a co-exist. A clear-cut division into the two groups was also found among the B19V strains from Dutch patients, and among the B19V sequences in GenBank. The two groups of genotype 1a co-exist around the world and do not appear to differ in their ability to cause disease. Strikingly, the two groups of B19V predominantly differ in synonymous mutations, distributed throughout the entire genome of B19V. We propose to call the two groups of B19V genotype 1a respectively subtype 1a1 and 1a2. PMID:22912828

  14. Identification of recombination in the NS1 and VPs genes of parvovirus B19.

    PubMed

    Shen, Hongxing; Zhang, Wen; Wang, Hua; Shao, Shihe

    2016-08-01

    Human parvovirus B19 (B19V), a member of the genus Erythrovirus of the family Parvoviridae, is a pathogenic virus distributed worldwide in the human population. In this study, we performed phylogenetic and recombination analysis of B19V based on the available nonstructural gene (NS1) and capsid proteins (VPs) genes in GenBank. Results indicated that recombination occurred between genotypes 3 and 1, leading to the recombinant cluster genotype 2. Other three inter-genotype recombination events were also discovered. Moreover, our results showed that among the four recombinant events in the present study, all of the major parents belonged to genotype 1, the minor parents were from genotypes 3 or 2, and all of the recombinants belonged to genotype 2. These recombinant events were confirmed by SimPlot Program and phylogenetic analysis. J. Med. Virol. 88:1457-1461, 2016. © 2016 Wiley Periodicals, Inc.

  15. Parvovirus-B19 and hematologic disorders.

    PubMed

    Yetgin, Sevgi; Aytaç Elmas, Selin

    2010-12-01

    Parvovirus-B19 (PV-B19) is a member of Parvoviridae, which is one of the smallest DNA viruses. PV-B19-associated diseases usually serve as a good representation of the balance of virus, host response and the immune system. The diseases manifested with PV-B19 are erythema infectiosum, which is common in children, hydrops fetalis, transient pure red cell aplasia in patients with chronic hemolytic anemia, arthralgia - mostly observed in women, and chronic pure red cell aplasia in immunocompromised individuals. Cytopenia (bicytopenia, monocytopenia or pancytopenia) may also accompany the diseases mentioned above. On the other hand, there are many diseases, including neurologic, vasculitic, hepatic, rheumatoid, nephritic, autoimmune, myocardial, and others in which the mechanisms of the diseases are not clear, which may be associated with PV-B19. The virus may manifest with unexpected and unexplained clinical pictures and lead to misdiagnosis. Therefore, hematologic disorders in any unestablished clinical diagnosis should be investigated for PV-B19 infection. However, serologic examination for PV-B19 diagnosis is not sufficient in immunocompromised status. The virus can be determined with polymerase chain reaction (PCR) in the serum or tissue samples. Supportive therapy, blood transfusion and immunoglobulin are the conventional therapeutic interventions for PV-B19 today. Vaccination studies are under examination. PMID:27263735

  16. Substitution rate and natural selection in parvovirus B19

    PubMed Central

    Stamenković, Gorana G.; Ćirković, Valentina S.; Šiljić, Marina M.; Blagojević, Jelena V.; Knežević, Aleksandra M.; Joksić, Ivana D.; Stanojević, Maja P.

    2016-01-01

    The aim of this study was to estimate substitution rate and imprints of natural selection on parvovirus B19 genotype 1. Studied datasets included 137 near complete coding B19 genomes (positions 665 to 4851) for phylogenetic and substitution rate analysis and 146 and 214 partial genomes for selection analyses in open reading frames ORF1 and ORF2, respectively, collected 1973–2012 and including 9 newly sequenced isolates from Serbia. Phylogenetic clustering assigned majority of studied isolates to G1A. Nucleotide substitution rate for total coding DNA was 1.03 (0.6–1.27) x 10−4 substitutions/site/year, with higher values for analyzed genome partitions. In spite of the highest evolutionary rate, VP2 codons were found to be under purifying selection with rare episodic positive selection, whereas codons under diversifying selection were found in the unique part of VP1, known to contain B19 immune epitopes important in persistent infection. Analyses of overlapping gene regions identified nucleotide positions under opposite selective pressure in different ORFs, suggesting complex evolutionary mechanisms of nucleotide changes in B19 viral genomes. PMID:27775080

  17. Investigation of human parvovirus B19 occurrence and genetic variability in different leukaemia entities.

    PubMed

    da Costa, A C; Bendit, I; de Oliveira, A C S; Kallas, E G; Sabino, E C; Sanabani, S S

    2013-01-01

    Human parvovirus B19V (B19V) has been associated with various haematological disorders, but data on its prevalence in leukaemia are scarce. In this cross-sectional study, we investigated patients in Sao Paulo, Brazil with leukaemia to determine the molecular frequency of B19 variants and characterize the viral genetic variability by partial and complete sequencing of the coding of non-structural protein 1 (NS1)/viral capsid proteins 1 and 2 (VP1/VP2). The presence of B19V infections was investigated by PCR amplification of the viral NS1 gene fragment and confirmed by sequencing analysis. The NS1/VP1/VP2 and partially larger gene fragments of the NS1-positive samples were determined by overlapping nested PCR and direct sequencing results. The B19V NS1 was detected in 40 (16%) of 249 bone marrow samples including 12/78 (15.4%) acute lymphoblastic leukaemia, 25/155 (16.1%) acute myeloid leukaemia and 3/16 (18.7%) chronic myeloid leukaemia samples. Of the 40 participants, 25 (62.5%) were infected with genotype 1a and 15 (37.5%) with genotype 3b. The phylogenetic analysis of other regions revealed that 12/40 (30%) of the patients with leukaemia were co-infected with genotypes 1a and 3b. In addition, a new B19V intergenotypic recombinant (1a/3b) and an NS1 non-recombinant genotype 1a were detected in one patient. Our findings demonstrated a relatively high prevalence of B19V monoinfections and dual infections and provide, for the first time, evidence of inter-genotypic recombination in adults with leukaemia that may contribute to the genetic diversity of B19V and may also be a source of new emerging viral strains with future implications for diagnosis, therapy and efficient vaccine development.

  18. Co-infection of human parvovirus B19 with Plasmodium falciparum contributes to malaria disease severity in Gabonese patients

    PubMed Central

    2013-01-01

    Background High seroprevalence of parvovirus B19 (B19V) coinfection with Plasmodium falciparum has been previously reported. However, the impact of B19V-infection on the clinical course of malaria is still elusive. In this study, we investigated the prevalence and clinical significance of B19V co-infection in Gabonese children with malaria. Methods B19V prevalence was analyzed in serum samples of 197 Gabonese children with P. falciparum malaria and 85 healthy controls using polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and direct DNA-sequencing. Results B19V was detected in 29/282 (10.28%) of Gabonese children. B19V was observed more frequently in P. falciparum malaria patients (14.21%) in comparison to healthy individuals (1.17%) (P<0.001). Notably, the mild-malaria group revealed significantly lower hematocrit levels in B19V/P. falciparum co-infection than in P. falciparum mono-infection (P<0.05). Genetic analysis revealed a predominance of B19V genotype-1 (71.43%) in the studied population. However, B19V-genotype 2 was observed significantly more often in children with severe-malaria than in mild-malaria (P=0.04). Conclusion Our findings reveal that B19V-infection is frequent in Gabonese children with P. falciparum malaria and signifies a possible contribution of B19V on the clinical course of malaria in a genotype-dependent manner. B19V co-infection should be considered as a additional diagnostic measure in malaria patients with life threatening anemia. PMID:23945350

  19. Parvovirus B19 Infection in Human Pregnancy

    PubMed Central

    Lamont, Ronald F.; Sobel, Jack; Vaisbuch, Edi; Kusanovic, Juan Pedro; Mazaki-Tovi, Shali; Kim, Sun Kwon; Uldbjerg, Niels; Romero, Roberto

    2010-01-01

    Human parvovirus B19 infection is widespread. Approximately 30-50% of pregnant women are non-immune and vertical transmission is common following maternal infection in pregnancy. Fetal infection may be associated with a normal outcome but fetal death may also occur without ultrasound evidence of infections sequelae. B19 infection should be considered in any case of non-immune hydrops. Diagnosis is mainly through serology and PCR. Surveillance requires sequential ultrasound and Doppler screening for signs of fetal anemia, heart failure, and hydrops. Immunoglobulins antiviral and vaccination are not yet available but intrauterine transfusion in selected cases can be lifesaving. PMID:21040396

  20. Parvovirus B19 in anemic liver transplant recipients.

    PubMed Central

    Ndimbie, O K; Frezza, E; Jordan, J A; Koch, W; van Thiel, D H

    1996-01-01

    Five hundred thirty-three liver transplant recipients were seen for follow-up care over a 6-month period. Of these, 23 (4.3%) had a hemoglobin level of < or = 9 g/dl, with 19 being eligible for inclusion in this study. The median hemoglobin level was 8.7 g/dl. Two patients had iron-deficiency anemia. All of the patients were on therapeutic drugs which can suppress erythropoiesis or shorten the lifespan of mature erythrocytes. Six patients (31.6%) were viremic for human parvovirus B19 but none was B19 immunoglobulin M seropositive. Two patients were immunoglobulin M seropositive for cytomegalovirus. The patients with circulating B19 DNA were not easily distinguished from those without the virus by their laboratory results. The absence of reticulocyte counts for these patients contributed to this inability to differentiate B19 from other causes of anemia, particularly drug myelotoxicity. The high likelihood of making a specific diagnosis with the increasing availability of PCR should spur the search for this virus in the liver transplant population. PMID:8914771

  1. Genetic drift of parvovirus B19 is found in AIDS patients with persistent B19 infection.

    PubMed

    Hung, Chien-Ching; Sheng, Wang-Hwei; Lee, Kuang-Lun; Yang, Shiu-Ju; Chen, Mao-Yuan

    2006-11-01

    It is generally thought that parvovirus B19 is stable genetically. Consistently, genetic drift has not been found in patients with persistent B19 infection. In this report, longitudinal genetic changes in NS1 and VP1 gene of B19 isolates from three AIDS patients with persistent B19 infection were studied. One of the three patients was not treated with highly active anti-retroviral therapy (HAART). B19 viral DNA from these patients was amplified by polymerase chain reaction (PCR) and then sequenced directly. A single genetic change was found in the B19 isolate obtained from the patient not treated with HAART on Day 10 after intravenous immunoglobulin (IVIG) treatment. The nucleotide sequences of B19 isolated from this patient, then remained unchanged over a period of 11 months. Analysis of NS1 clones derived from his longitudinal viral isolates showed the existence of quasi-species but genetic drift was not found. One of the other two patients treated with HAART experienced treatment failure; he was later treated with mega-HAART. In contrast to the genetic stability of B19 isolates from the patient not treated with HAART, multiple genetic changes were discovered in the viral isolates from the two other patients after HAART and mega-HAART, respectively. Through analysis of B19 clones, the frequency of clones containing these mutations confirmed the genetic drift. Nucleotide substitutions seen in VP2 gene of isolates with genetic drift from both patients were all non-conserved, suggesting that they are positively selected.

  2. Human parvovirus B19: a mechanistic overview of infection and DNA replication

    PubMed Central

    Luo, Yong; Qiu, Jianming

    2015-01-01

    Human parvovirus B19 (B19V) is a human pathogen that belongs to genus Erythroparvovirus of the Parvoviridae family, which is composed of a group of small DNA viruses with a linear single-stranded DNA genome. B19V mainly infects human erythroid progenitor cells and causes mild to severe hematological disorders in patients. However, recent clinical studies indicate that B19V also infects nonerythroid lineage cells, such as myocardial endothelial cells, and may be associated with other disease outcomes. Several cell culture systems, including permissive and semipermissive erythroid lineage cells, nonpermissive human embryonic kidney 293 cells and recently reported myocardial endothelial cells, have been used to study the mechanisms underlying B19V infection and B19V DNA replication. This review aims to summarize recent advances in B19V studies with a focus on the mechanisms of B19V tropism specific to different cell types and the cellular pathways involved in B19V DNA replication including cellular signaling transduction and cell cycle arrest. PMID:26097496

  3. Parvovirus B19 Infection of Human Primary Erythroid Progenitor Cells Triggers ATR-Chk1 Signaling, Which Promotes B19 Virus Replication ▿

    PubMed Central

    Luo, Yong; Lou, Sai; Deng, Xuefeng; Liu, Zhengwen; Li, Yi; Kleiboeker, Steve; Qiu, Jianming

    2011-01-01

    Human parvovirus B19 (B19V) infection is restricted to erythroid progenitor cells of the human bone marrow. Although the mechanism by which the B19V genome replicates in these cells has not been studied in great detail, accumulating evidence has implicated involvement of the cellular DNA damage machinery in this process. Here, we report that, in ex vivo-expanded human erythroid progenitor cells, B19V infection induces a broad range of DNA damage responses by triggering phosphorylation of all the upstream kinases of each of three repair pathways: ATM (ataxia-telangiectasi mutated), ATR (ATM and Rad3 related), and DNA-PKcs (DNA-dependent protein kinase catalytic subunit). We found that phosphorylated ATM, ATR, and DNA-PKcs, and also their downstream substrates and components (Chk2, Chk1, and Ku70/Ku80 complex, respectively), localized within the B19V replication center. Notably, inhibition of kinase phosphorylation (through treatment with either kinase-specific inhibitors or kinase-specific shRNAs) revealed requirements for signaling of ATR and DNA-PKcs, but not ATM, in virus replication. Inhibition of the ATR substrate Chk1 led to similar levels of decreased virus replication, indicating that signaling via the ATR-Chk1 pathway is critical to B19V replication. Notably, the cell cycle arrest characteristic of B19V infection was not rescued by interference with the activity of any of the three repair pathway kinases. PMID:21680529

  4. Classification of hepatitis B virus genotypes by the PCR-Invader method with genotype-specific probes.

    PubMed

    Tadokoro, Kenichi; Kobayashi, Mariko; Yamaguchi, Toshikazu; Suzuki, Fumitaka; Miyauchi, Saeko; Egashira, Toru; Kumada, Hiromitsu

    2006-12-01

    Hepatitis B virus is a worldwide public health problem. A simple and effective test to identify viral genotypes would greatly aid efforts to understand and control the spread of this disease. A serial invasive signal amplification reaction assay (PCR-Invader assay) was developed for distinguishing the known eight genotypes (A-H) and four subgenotypes (Aa, Ae, Ba, Bj) of hepatitis B virus (HBV). The preS/S and core regions were amplified by multiplex PCR and delivered to 12 wells containing genotype-specific Invader probes. By observing the fluorescence patterns in the wells, HBV sub/genotypes can be assigned. A total of 505 serum samples containing HBV/HBsAg in Japan was examined by PCR-Invader and compared the results with those from ELISA assays with monoclonal antibodies against epitopes on gene products of the preS2 region and with a genotype-specific probe assay (GSPA) based on the preS1 region. Genotypes determined by the PCR-Invader agreed with those of the ELISA method in 98.2% of cases and with the GSPA method in 97.1% of cases. Co-infection with two distinct genotypes was correctly identified by the PCR-Invader in four serum samples, as determined by GSPA. Thus, the PCR-Invader assay is a useful tool for detecting the 10 known HBV sub/genotypes. PMID:16934340

  5. Antibody-mediated opsonization of red blood cells in parvovirus B19 infection.

    PubMed

    Chehadeh, Wassim; Halim, Medhat A; Al-Nakib, Widad

    2009-07-20

    Red blood cells (RBCs) express abundantly parvovirus B19 receptor, and their role in the dissemination or clearance of B19 infection is unknown. In this study, we report that in early, acute or persistent infection, B19 viremia is mostly associated with RBCs. The capacity of different patients' plasma or IgG to opsonize RBCs collected from patients with early B19 infection, was investigated. The highest opsonization activity was observed with plasma or IgG fractions from patients with past B19 infection. In contrast, IgG samples from patients with acute or persistent infection showed no or little opsonization activity. The depletion of antibodies specific to B19 VP1, but not VP2, from IgG samples, resulted in a significant suppression of opsonization. Furthermore, IgG samples preincubated with heated B19 particles exposing VP1-unique (VP1u) region were unable to opsonize RBCs. These observations clearly suggest a role for anti-VP1u IgG in the opsonization of RBC-bound B19 particles.

  6. Hepatitis C virus infection: Are there still specific problems with genotype 3?

    PubMed Central

    Gondeau, Claire; Pageaux, Georges Philippe; Larrey, Dominique

    2015-01-01

    Hepatitis C virus (HCV) infection is one of the most common causes of chronic liver disease and the main indication for liver transplantation worldwide. As promising specific treatments have been introduced for genotype 1, clinicians and researchers are now focusing on patients infected by non-genotype 1 HCV, particularly genotype 3. Indeed, in the golden era of direct-acting antiviral drugs, genotype 3 infections are no longer considered as easy to treat and are associated with higher risk of developing severe liver injuries, such as cirrhosis and hepatocellular carcinoma. Moreover, HCV genotype 3 accounts for 40% of all HCV infections in Asia and is the most frequent genotype among HCV-positive injecting drug users in several countries. Here, we review recent data on HCV genotype 3 infection/treatment, including clinical aspects and the underlying genotype-specific molecular mechanisms. PMID:26576095

  7. Human parvovirus B19 surveillance in patients with rash and fever from Belarus.

    PubMed

    Yermalovich, Marina A; Hübschen, Judith M; Semeiko, Galina V; Samoilovich, Elena O; Muller, Claude P

    2012-06-01

    Human parvovirus B19 (B19V) infection in immunocompetent patients usually has a mild clinical course, but during pregnancy it can cause serious and even fatal complications in the fetus. The most common clinical presentation of B19V infection is erythema infectiosum and in this case laboratory confirmation is required for differentiation from other exanthematous diseases. Measles and rubella negative sera collected in Belarus between 2005 and 2008 from 906 patients with a rash and fever were screened for B19V infection by ELISA. More than 35% of the samples (322/906) were positive for B19V. The proportion ranged from 10.1% in 2008 to 53.2% in 2006 when an outbreak took place in Minsk city. All B19V outbreaks and cluster cases occurred during the winter-spring period, but sporadic cases were recorded basically throughout the year. The majority of the cases (56.5%) occurred among the 2 till 10 year old children, and 27.3% of the cases were observed in adults between 19 and 53 years. All 104 B19V strains sequenced in the NS1/VP1u region belonged to genotype 1 with a maximal genetic distance of 1.75%. The two phylogenetic clusters reflected the geographic origins of the viruses within the country. Forty-two unique nucleotide mutations as compared to sequences downloaded from GenBank were found in the VP1u and NS1 regions; most of these changes were nonsynonymous. This report highlights the importance of B19V infection in patients with a rash and fever in Belarus.

  8. Human parvovirus B19 VP2 empty capsids bind to human villous trophoblast cells in vitro via the globoside receptor.

    PubMed Central

    Wegner, Carole C; Jordan, Jeanne A

    2004-01-01

    BACKGROUND: Pregnant women acutely infected with human parvovirus B19 (B19) may transmit the virus to the developing fetus. The mechanism whereby the virus interacts with the placenta is unknown. It is known that globoside receptor is required for successful infection of the target cells, which are the highly undifferentiated, actively dividing colony and burst-form units of the erythroid series. Globoside is present on trophoblast cells which have intimate contact with maternal blood, and may therefore serve as a potential route for B19 transmission into the fetal compartment. OBJECTIVES: The purpose of this study was to determine whether B19 VP2 capsids could bind to villous trophoblast cells in vitro and whether globoside was involved. METHODS: Binding of B19 VP2 empty capsid to first-trimester villous trophoblast cells was assessed by multiple approaches, including ICC using either biotinylated B19 VP2 empty capsid or unlabeled B19 VP2 empty capsid. Quantification of viral binding involved I125-labeled B19 VP2 empty capsid. Competition studies included excess unlabeled empty capsids or pretreatment with globoside-specific IgM antibody. RESULTS: Linear binding of B19 VP2 capsid to purified villous trophoblast cells in vitro was clearly demonstrated (R2= 0.9524). Competition studies revealed specificity of I125-labeled B19 VP2 capsid binding to villous trophoblast cells when pretreatment with either 60-fold excess unlabeled B19 capsid or globoside-specific IgM antibody took place. The results illustrated B19's ability to bind in a specific manner to globoside-containing villous trophoblast cells. CONCLUSION: We speculate that the globoside present on trophoblast cells may play a role in viral binding in vivo, which may facilitate B19 transmission across the maternal-fetal interface. PMID:15739820

  9. Comparative proteomics lends insight into genotype-specific pathogenicity.

    PubMed

    Guarnieri, Michael T

    2013-09-01

    Comparative proteomic analyses have emerged as a powerful tool for the identification of unique biomarkers and mechanisms of pathogenesis. In this issue of Proteomics, Murugaiyan et al. utilize difference gel electrophoresis (DIGE) to examine differential protein expression between nonpathogenic and pathogenic genotypes of Prototheca zopfii, a causative agent in bovine enteritis and mastitis. Their findings provide insights into molecular mechanisms of infection and evolutionary adaptation of pathogenic genotypes, demonstrating the power of comparative proteomic analyses. PMID:23925996

  10. The VP1u Receptor Restricts Parvovirus B19 Uptake to Permissive Erythroid Cells

    PubMed Central

    Leisi, Remo; Von Nordheim, Marcus; Ros, Carlos; Kempf, Christoph

    2016-01-01

    Parvovirus B19 (B19V) is a small non-enveloped virus and known as the causative agent for the mild childhood disease erythema infectiosum. B19V has an extraordinary narrow tissue tropism, showing only productive infection in erythroid precursor cells in the bone marrow. We recently found that the viral protein 1 unique region (VP1u) contains an N-terminal receptor-binding domain (RBD), which mediates the uptake of the virus into cells of the erythroid lineage. To further investigate the role of the RBD in connection with a B19V-unrelated capsid, we chemically coupled the VP1u of B19V to the bacteriophage MS2 capsid and tested the internalization capacity of the bioconjugate on permissive cells. In comparison, we studied the cellular uptake and infection of B19V along the erythroid differentiation. The results showed that the MS2-VP1u bioconjugate mimicked the specific internalization of the native B19V into erythroid precursor cells, which further coincides with the restricted infection profile. The successful mimicry of B19V uptake demonstrates that the RBD in the VP1u is sufficient for the endocytosis of the viral capsid. Furthermore, the recombinant VP1u competed with B19V uptake into permissive cells, thus excluding a significant alternative uptake mechanism by other receptors. Strikingly, the VP1u receptor appeared to be expressed only on erythropoietin-dependent erythroid differentiation stages that also provide the necessary intracellular factors for a productive infection. Taken together, these findings suggest that the VP1u binds to a yet-unknown erythroid-specific cellular receptor and thus restricts the virus entry to permissive cells. PMID:27690083

  11. Amazonian head lice-specific genotypes are putatively pre-Columbian.

    PubMed

    Boutellis, Amina; Veracx, Aurélie; Abrahão, Jônatas; Raoult, Didier

    2013-06-01

    Head and body lice are strict obligate human ectoparasites with three mitochondrial phylotypes (A, B, and C). Using molecular methods for genotyping lice (Cytochrome b and multi-spacer typing), and comparing our results with all the sequences of human lice that were genotyped previously, we assessed the presence of a specific American genotype that most likely predates the Columbian era in head lice collected from Amazonia. PMID:23610158

  12. Original Research: Parvovirus B19 infection in children with sickle cell disease in the hydroxyurea era

    PubMed Central

    Penkert, Rhiannon R; Lavoie, Paul; Tang, Li; Sun, Yilun; Hurwitz, Julia L

    2016-01-01

    Parvovirus B19 infection causes transient aplastic crisis in sickle cell disease (SCD) due to a temporary interruption in the red blood cell production. Toxicity from hydroxyurea includes anemia and reticulocytopenia, both of which also occur during a transient aplastic crisis event. Hydroxyurea inhibits proliferation of hematopoietic cells and may be immunosuppressive. We postulated that hydroxyurea could exacerbate parvovirus B19-induced aplastic crisis and inhibit the development of specific immune responses in children with SCD. We conducted a retrospective review of parvovirus B19 infection in 330 children with SCD. Altogether there were 120 known cases of aplastic crisis attributed to parvovirus B19 infection, and 12% of children were on hydroxyurea treatment during the episode. We evaluated hematological and immune responses. Children with HbSS or HbSβ0-thalassemia treated with hydroxyurea, when compared with untreated children, required fewer transfusions and had higher Hb concentration nadir during transient aplastic crisis. Duration of hospital stays was no different between hydroxyurea-treated and untreated groups. Children tested within a week following aplastic crisis were positive for parvovirus-specific IgG. Immune responses lasted for the duration of the observation period, up to 13 years after transient aplastic crisis, and there were no repeat aplastic crisis episodes. The frequencies of parvovirus-specific antibodies in all children with SCD increased with age, as expected due to the increased likelihood of a parvovirus exposure, and were comparable to frequencies reported for healthy children. Approximately one-third of children had a positive parvovirus B19-specific IgG test without a documented history of transient aplastic crisis, and 64% of them were treated with hydroxyurea. Hydroxyurea may reduce requirements for blood transfusions and may attenuate symptoms during transient aplastic crisis episodes caused by parvovirus B19 infections

  13. The small 11kDa nonstructural protein of human parvovirus B19 plays a key role in inducing apoptosis during B19 virus infection of primary erythroid progenitor cells

    PubMed Central

    Chen, Aaron Yun; Zhang, Elizabeth Yan; Guan, Wuxiang; Cheng, Fang; Kleiboeker, Steve; Yankee, Thomas M.

    2010-01-01

    Human parvovirus B19 (B19V) infection shows a strong erythroid tropism and drastically destroys erythroid progenitor cells, thus leading to most of the disease outcomes associated with B19V infection. In this study, we systematically examined the 3 B19V nonstructural proteins, 7.5kDa, 11kDa, and NS1, for their function in inducing apoptosis in transfection of primary ex vivo–expanded erythroid progenitor cells, in comparison with apoptosis induced during B19V infection. Our results show that 11kDa is a more significant inducer of apoptosis than NS1, whereas 7.5kDa does not induce apoptosis. Furthermore, we determined that caspase-10, an initiator caspase in death receptor signaling, is the most active caspase in apoptotic erythroid progenitors induced by 11kDa and NS1 as well as during B19V infection. More importantly, cytoplasm-localized 11kDa is expressed at least 100 times more than nucleus-localized NS1 at the protein level in primary erythroid progenitor cells infected with B19V; and inhibition of 11kDa expression using antisense oligos targeting specifically to the 11kDa-encoding mRNAs reduces apoptosis significantly during B19V infection of erythroid progenitor cells. Taken together, these results demonstrate that the 11kDa protein contributes to erythroid progenitor cell death during B19V infection. PMID:19861680

  14. Specific HLA genotypes confer susceptibility to acute necrotizing encephalopathy.

    PubMed

    Hoshino, A; Saitoh, M; Miyagawa, T; Kubota, M; Takanashi, J-I; Miyamoto, A; Tokunaga, K; Oka, A; Mizuguchi, M

    2016-09-01

    Acute necrotizing encephalopathy (ANE) is a rare and severe syndrome of acute encephalopathy triggered by viral infections. Cytokine storm is considered as the main pathogenetic mechanism of ANE. ANE is prevalent in East Asia, suggesting the association of host genetic factors. To elucidate the genetic background of Japanese ANE, we examined genotypes of human leukocyte antigen (HLA)-A, C, B, DRB1, DQB1 and DPB1 in 31 patients. Significant positive association was observed in both the allele frequency and positivity of DRB1*09:01 (P=0.043 and 0.025, respectively), as well as those of DQB1*03:03 (P=0.034 and 0.026, respectively). The carrier frequency of DRB1*09:01 and DQB1*03:03 alleles was higher in the patients (45.16%) than in controls (28.57%). These alleles are more common in East Asian than in European populations, and are reportedly associated with various autoimmune diseases in Japanese patients. Our data provide further evidence that altered immune response based on individual HLA genotypes may contribute to ANE pathogenesis. PMID:27467284

  15. Microarrays for Genotyping Human Group A Rotavirus by Multiplex Capture and Type-Specific Primer Extension

    PubMed Central

    Lovmar, Lovisa; Fock, Caroline; Espinoza, Felix; Bucardo, Filemon; Syvänen, Ann-Christine; Bondeson, Kåre

    2003-01-01

    Human group A rotavirus (HRV) is the major cause of severe gastroenteritis in infants worldwide. HRV shares the feature of a high degree of genetic diversity with many other RNA viruses, and therefore, genotyping of this organism is more complicated than genotyping of more stable DNA viruses. We describe a novel microarray-based method that allows high-throughput genotyping of RNA viruses with a high degree of polymorphism by multiplex capture and type-specific extension on microarrays. Denatured reverse transcription (RT)-PCR products derived from two outer capsid genes of clinical isolates of HRV were hybridized to immobilized capture oligonucleotides representing the most commonly occurring P and G genotypes on a microarray. Specific primer extension of the type-specific capture oligonucleotides was applied to incorporate the fluorescent nucleotide analogue cyanine 5-labeled dUTP as a detectable label. Laser scanning and fluorescence detection of the microarrays was followed by visual or computer-assisted interpretation of the fluorescence patterns generated on the microarrays. Initially, the method detected HRV in all 40 samples and correctly determined both the G and the P genotypes of 35 of the 40 strains analyzed. After modification by inclusion of additional capture oligonucleotides specific for the initially unassigned genotypes, all genotypes could be correctly defined. The results of genotyping with the microarray fully agreed with the results obtained by nucleotide sequence analysis and sequence-specific multiplex RT-PCR. Owing to its robustness, simplicity, and general utility, the microarray-based method may gain wide applicability for the genotyping of microorganisms, including highly variable RNA and DNA viruses. PMID:14605152

  16. Microarrays for genotyping human group a rotavirus by multiplex capture and type-specific primer extension.

    PubMed

    Lovmar, Lovisa; Fock, Caroline; Espinoza, Felix; Bucardo, Filemon; Syvänen, Ann-Christine; Bondeson, Kåre

    2003-11-01

    Human group A rotavirus (HRV) is the major cause of severe gastroenteritis in infants worldwide. HRV shares the feature of a high degree of genetic diversity with many other RNA viruses, and therefore, genotyping of this organism is more complicated than genotyping of more stable DNA viruses. We describe a novel microarray-based method that allows high-throughput genotyping of RNA viruses with a high degree of polymorphism by multiplex capture and type-specific extension on microarrays. Denatured reverse transcription (RT)-PCR products derived from two outer capsid genes of clinical isolates of HRV were hybridized to immobilized capture oligonucleotides representing the most commonly occurring P and G genotypes on a microarray. Specific primer extension of the type-specific capture oligonucleotides was applied to incorporate the fluorescent nucleotide analogue cyanine 5-labeled dUTP as a detectable label. Laser scanning and fluorescence detection of the microarrays was followed by visual or computer-assisted interpretation of the fluorescence patterns generated on the microarrays. Initially, the method detected HRV in all 40 samples and correctly determined both the G and the P genotypes of 35 of the 40 strains analyzed. After modification by inclusion of additional capture oligonucleotides specific for the initially unassigned genotypes, all genotypes could be correctly defined. The results of genotyping with the microarray fully agreed with the results obtained by nucleotide sequence analysis and sequence-specific multiplex RT-PCR. Owing to its robustness, simplicity, and general utility, the microarray-based method may gain wide applicability for the genotyping of microorganisms, including highly variable RNA and DNA viruses.

  17. Host specificity and source of Enterocytozoon bieneusi genotypes in a drinking source watershed.

    PubMed

    Guo, Yaqiong; Alderisio, Kerri A; Yang, Wenli; Cama, Vitaliano; Feng, Yaoyu; Xiao, Lihua

    2014-01-01

    To assess the host specificity of Enterocytozoon bieneusi and to track the sources of E. bieneusi contamination, we genotyped E. bieneusi in wildlife and stormwater from the watershed of New York City's source water, using ribosomal internal transcribed spacer (ITS)-based PCR and sequence analyses. A total of 255 specimens from 23 species of wild mammals and 67 samples from stormwater were analyzed. Seventy-four (29.0%) of the wildlife specimens and 39 (58.2%) of the stormwater samples from streams were PCR positive. Altogether, 20 E. bieneusi genotypes were found, including 8 known genotypes and 12 new ones. Sixteen and five of the genotypes were seen in animals and stormwater from the watershed, respectively, with WL4 being the most common genotype in both animals (35 samples) and stormwater (23 samples). The 20 E. bieneusi genotypes belonged to five genogroups (groups 1, 3, 4, and 7 and an outlier), with only 23/113 (20.4%) E. bieneusi-positive samples belonging to zoonotic genogroup 1 and 3/20 genotypes ever being detected in humans. The two genogroups previously considered host specific, groups 3 and 4, were both detected in multiple groups of mammals. Thus, with the exception of the type IV, Peru11, and D genotypes, which were detected in only 7, 5, and 2 animals, respectively, most E. bieneusi strains in most wildlife samples and all stormwater samples in the watershed had no known public health significance, as these types have not previously been detected in humans. The role of different species of wild mammals in the contribution of E. bieneusi contamination in stormwater was supported by determinations of host-adapted Cryptosporidium species/genotypes in the same water samples. Data from this study indicate that the host specificity of E. bieneusi group 3 is broader than originally thought, and wildlife is the main source of E. bieneusi in stormwater in the watershed.

  18. Study of Non–organ-specific Antibodies in Children with Genotype 4 Chronic Hepatitis C

    PubMed Central

    Hamed, Mohammed E.; Alanani, Naglaa M. Kamal; Sherief, Laila M.; Fouad, Mohammed A.; Elwahab, Lamiaa A.; Raafat, Nermin

    2013-01-01

    Background/Aim: Adult studies established a relationship between hepatitis C virus (HCV) infection and the presence of non–organ-specific antibodies (NOSAs). Most studies were carried out on genotypes 1 and 2. Only a few studies addressed that issue in pediatrics. No studies have been carried out on autoimmunity and genotype 4 in children. We aim to investigate NOSAs in 80 Egyptian children with chronic HCV infection along with studying the underlying genotype of HCV, and correlating autoimmunity with the epidemiological, clinical, biochemical, and virological features. Materials and Methods: HCV-RNA was assayed by the polymerase chain reaction and viral genotypes were determined. NOSAs were measured and liver biopsies were taken for histopathological examination. Results: Genotype4 was the only detected genotype in the included 80 patients. Anti-smooth muscle antibodies (ASMA) were the only detected antibodies in 32 (40%) patients, always with V specificity (vessels only) at titers ranging from 1:20 and 1:160. Anti-nuclear antibodies (ANA) and liver–kidney microsomal antibodies-1 (LKMA-1) were not detected in any of our patients. Epidemiologic and clinical features did not significantly differ between autoantibody-positive and -negative patients. Among biochemical features, significantly high levels of total bilirubin, albumin, immunoglobulins, alkaline phosphatase, and gamma-glutamyl transpeptidase were found in the antibody-positive group. Conclusion: Genotype 4 HCV is the prevailing genotype in Egyptian children with chronic HCV infection. A consistent proportion of these children with chronic HCV infection circulate non–organ-specific autoantibodies. The prevalence of ASMA and the absence of ANA and LKMA-1 might be related to the unique situation in Egypt with unique prevalence of genotype 4. More studies are warranted on larger pediatric population to validate these findings. PMID:24195980

  19. Host Specificity and Source of Enterocytozoon bieneusi Genotypes in a Drinking Source Watershed

    PubMed Central

    Guo, Yaqiong; Alderisio, Kerri A.; Yang, Wenli; Cama, Vitaliano; Xiao, Lihua

    2014-01-01

    To assess the host specificity of Enterocytozoon bieneusi and to track the sources of E. bieneusi contamination, we genotyped E. bieneusi in wildlife and stormwater from the watershed of New York City's source water, using ribosomal internal transcribed spacer (ITS)-based PCR and sequence analyses. A total of 255 specimens from 23 species of wild mammals and 67 samples from stormwater were analyzed. Seventy-four (29.0%) of the wildlife specimens and 39 (58.2%) of the stormwater samples from streams were PCR positive. Altogether, 20 E. bieneusi genotypes were found, including 8 known genotypes and 12 new ones. Sixteen and five of the genotypes were seen in animals and stormwater from the watershed, respectively, with WL4 being the most common genotype in both animals (35 samples) and stormwater (23 samples). The 20 E. bieneusi genotypes belonged to five genogroups (groups 1, 3, 4, and 7 and an outlier), with only 23/113 (20.4%) E. bieneusi-positive samples belonging to zoonotic genogroup 1 and 3/20 genotypes ever being detected in humans. The two genogroups previously considered host specific, groups 3 and 4, were both detected in multiple groups of mammals. Thus, with the exception of the type IV, Peru11, and D genotypes, which were detected in only 7, 5, and 2 animals, respectively, most E. bieneusi strains in most wildlife samples and all stormwater samples in the watershed had no known public health significance, as these types have not previously been detected in humans. The role of different species of wild mammals in the contribution of E. bieneusi contamination in stormwater was supported by determinations of host-adapted Cryptosporidium species/genotypes in the same water samples. Data from this study indicate that the host specificity of E. bieneusi group 3 is broader than originally thought, and wildlife is the main source of E. bieneusi in stormwater in the watershed. PMID:24141128

  20. Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping.

    PubMed

    Lee, Han B; Schwab, Tanya L; Koleilat, Alaa; Ata, Hirotaka; Daby, Camden L; Cervera, Roberto Lopez; McNulty, Melissa S; Bostwick, Hannah S; Clark, Karl J

    2016-06-01

    Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98-100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score

  1. Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping.

    PubMed

    Lee, Han B; Schwab, Tanya L; Koleilat, Alaa; Ata, Hirotaka; Daby, Camden L; Cervera, Roberto Lopez; McNulty, Melissa S; Bostwick, Hannah S; Clark, Karl J

    2016-06-01

    Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98-100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score

  2. Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping

    PubMed Central

    Lee, Han B.; Schwab, Tanya L.; Koleilat, Alaa; Ata, Hirotaka; Daby, Camden L.; Cervera, Roberto Lopez; McNulty, Melissa S.; Bostwick, Hannah S.; Clark, Karl J.

    2016-01-01

    Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98–100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score

  3. Detection of human parvovirus B19 in papillary thyroid carcinoma

    PubMed Central

    Wang, J H; Zhang, W P; Liu, H X; Wang, D; Li, Y F; Wang, W Q; Wang, L; He, F R; Wang, Z; Yan, Q G; Chen, L W; Huang, G S

    2008-01-01

    To evaluate whether parvovirus B19, a common human pathogen, was also involved in papillary thyroid carcinoma (PTC), 112 paraffin-embedded thyroid specimens of benign nodules, papillary, medullary and follicular carcinomas, and normal controls were examined for B19 DNA and capsid protein by nested PCR, in situ hybridisation (ISH) and immunohistochemistry (IHC). The expression of the nuclear factor-κB (NF-κB) was investigated by IHC. The results showed B19 DNA commonly exists in human thyroid tissues; however, there were significant differences between PTC group and normal controls, and between PTC and nonneoplastic adjacent tissues (P<0.001). The presence of viral DNA in PTC neoplastic epithelium was confirmed by laser-capture microdissection and sequencing of nested PCR products. B19 capsid protein in PTC group was significantly higher than that of all the control groups and nonneoplastic adjacent tissues (P⩽0.001). Compared with control groups, the activation of NF-κB in PTC group was significantly increased (P⩽0.02), except for medullary carcinomas, and the activation of NF-κB was correlated with the viral protein presence (P=0.002). Moreover, NF-κB was colocalised with B19 DNA in the neoplastic epithelium of PTC by double staining of IHC and ISH. These results indicate for the first time a possible role of B19 in pathogenesis of PTC. PMID:18212749

  4. S-genotype identification based on allele-specific PCR in Japanese pear

    PubMed Central

    Nashima, Kenji; Terakami, Shingo; Nishio, Sogo; Kunihisa, Miyuki; Nishitani, Chikako; Saito, Toshihiro; Yamamoto, Toshiya

    2015-01-01

    Gametophytic self-incompatibility in Japanese pear (Pyrus pyrifolia Nakai) is controlled by the single, multi-allelic S-locus. Information about the S-genotypes is important for breeding and the selection of pollen donors for fruit production. Rapid and reliable S-genotype identification system is necessary for efficient breeding of new cultivars in Japanese pear. We designed S allele-specific PCR primer pairs for ten previously reported S-RNase alleles (S1–S9 and Sk) as simple and reliable method. Specific nucleotide sequences were chosen to design the primers to amplify fragments of only the corresponding S alleles. The developed primer pairs were evaluated by using homozygous S-genotypes (S1/S1–S9/S9 and S4sm/S4sm) and 14 major Japanese pear cultivars, and found that S allele-specific primer pairs can identify S-genotypes effectively. The S allele-specific primer pairs developed in this study will be useful for efficient S-genotyping and for marker-assisted selection in Japanese pear breeding programs. PMID:26175617

  5. Parvovirus B19 Replication and Expression in Differentiating Erythroid Progenitor Cells

    PubMed Central

    Bua, Gloria; Manaresi, Elisabetta; Bonvicini, Francesca; Gallinella, Giorgio

    2016-01-01

    The pathogenic Parvovirus B19 (B19V) is characterized by a strict adaptation to erythroid progenitor cells (EPCs), a heterogeneous population of differentiating cells with diverse phenotypic and functional properties. In our work, we studied the dynamics of B19V infection in EPCs in dependence on the cell differentiation stage, in terms of distribution of infected cells, synthesis of viral nucleic acids and production of infectious virus. EPCs at early differentiation stage led to an abortive infection, without viral genome replication and a very low transcriptional activity. EPCs at later stages were permissive, with highest levels of viral replicative activity at day 9 (+3.0 Log from 2 to 48 hpi) and lower levels at day 18 (+1.5 Log from 2 to 48 hpi). B19V DNA increment was in accordance with the percentage of cells positive to flow-FISH assay (41.4% at day 9, 1.1% at day 18). Quantitation of total RNA indicated a close association of genome replication and transcription with viral RNA accumulation within infected cells related to viral DNA increase during the course of infection. Analysis of the different classes of mRNAs revealed two distinct pattern of genome expression profile with a fine regulation in the frequency utilization of RNA processing signals: an early phase, when cleavage at the proximal site leading to a higher relative production of mRNA for NS protein, and a late phase, when cleavage at the distal site was more frequent leading to higher relative abundance of mRNA for VP and 11 kDA proteins. Infectious virus was released from cells at day 6–15, but not at day 18. Our results, providing a detailed description of B19V replication and expression profile in differentiating EPCs, highlight the very tight adaptation of B19V to a specific cellular target defined both by its erythroid lineage and its differentiation stage. PMID:26845771

  6. The Receptor-Binding Domain in the VP1u Region of Parvovirus B19.

    PubMed

    Leisi, Remo; Di Tommaso, Chiarina; Kempf, Christoph; Ros, Carlos

    2016-03-01

    Parvovirus B19 (B19V) is known as the human pathogen causing the mild childhood disease erythema infectiosum. B19V shows an extraordinary narrow tissue tropism for erythroid progenitor cells in the bone marrow, which is determined by a highly restricted uptake. We have previously shown that the specific internalization is mediated by the interaction of the viral protein 1 unique region (VP1u) with a yet unknown cellular receptor. To locate the receptor-binding domain (RBD) within the VP1u, we analyzed the effect of truncations and mutations on the internalization capacity of the recombinant protein into UT7/Epo cells. Here we report that the N-terminal amino acids 5-80 of the VP1u are necessary and sufficient for cellular binding and internalization; thus, this N-terminal region represents the RBD required for B19V uptake. Using site-directed mutagenesis, we further identified a cluster of important amino acids playing a critical role in VP1u internalization. In silico predictions and experimental results suggest that the RBD is structured as a rigid fold of three α-helices. Finally, we found that dimerization of the VP1u leads to a considerably enhanced cellular binding and internalization. Taken together, we identified the RBD that mediates B19V uptake and mapped functional and structural motifs within this sequence. The findings reveal insights into the uptake process of B19V, which contribute to understand the pathogenesis of the infection and the neutralization of the virus by the immune system.

  7. The Receptor-Binding Domain in the VP1u Region of Parvovirus B19

    PubMed Central

    Leisi, Remo; Di Tommaso, Chiarina; Kempf, Christoph; Ros, Carlos

    2016-01-01

    Parvovirus B19 (B19V) is known as the human pathogen causing the mild childhood disease erythema infectiosum. B19V shows an extraordinary narrow tissue tropism for erythroid progenitor cells in the bone marrow, which is determined by a highly restricted uptake. We have previously shown that the specific internalization is mediated by the interaction of the viral protein 1 unique region (VP1u) with a yet unknown cellular receptor. To locate the receptor-binding domain (RBD) within the VP1u, we analyzed the effect of truncations and mutations on the internalization capacity of the recombinant protein into UT7/Epo cells. Here we report that the N-terminal amino acids 5–80 of the VP1u are necessary and sufficient for cellular binding and internalization; thus, this N-terminal region represents the RBD required for B19V uptake. Using site-directed mutagenesis, we further identified a cluster of important amino acids playing a critical role in VP1u internalization. In silico predictions and experimental results suggest that the RBD is structured as a rigid fold of three α-helices. Finally, we found that dimerization of the VP1u leads to a considerably enhanced cellular binding and internalization. Taken together, we identified the RBD that mediates B19V uptake and mapped functional and structural motifs within this sequence. The findings reveal insights into the uptake process of B19V, which contribute to understand the pathogenesis of the infection and the neutralization of the virus by the immune system. PMID:26927158

  8. Genotype-specific risk stratification and management of patients with long QT syndrome.

    PubMed

    Barsheshet, Alon; Dotsenko, Olena; Goldenberg, Ilan

    2013-11-01

    Long QT syndrome (LQTS) is an inherited disorder associated with life-threatening ventricular arrhythmias. An understanding of the relationship between the genotype and phenotype characteristics of LQTS can lead to improved risk stratification and management of this hereditary arrhythmogenic disorder. Risk stratification in LQTS relies on combined assessment of clinical, electrocardiographic, and mutations-specific factors. Studies have shown that there are genotype-specific risk factors for arrhythmic events including age, gender, resting heart rate, QT corrected for heart rate, prior syncope, the postpartum period, menopause, mutation location, type of mutation, the biophysical function of the mutation, and response to beta-blockers. Importantly, genotype-specific therapeutic options have been suggested. Lifestyle changes are recommended according to the prevalent trigger for cardiac events. Beta-blockers confer greater benefit among patients with LQT1 with the greatest benefit among those with cytoplasmic loops mutations; specific beta-blocker agents may provide greater protection than other agents in specific LQTS genotypes. Potassium supplementation and sex hormone-based therapy may protect patients with LQT2. Sodium channel blockers such as mexiletine, flecainide, and ranolazine could be treatment options in LQT3. PMID:24206565

  9. Antibody-Mediated Enhancement of Parvovirus B19 Uptake into Endothelial Cells Mediated by a Receptor for Complement Factor C1q

    PubMed Central

    von Kietzell, Kristina; Pozzuto, Tanja; Heilbronn, Regine; Grössl, Tobias; Fechner, Henry

    2014-01-01

    ABSTRACT Despite its strong host tropism for erythroid progenitor cells, human parvovirus B19 (B19V) can also infect a variety of additional cell types. Acute and chronic inflammatory cardiomyopathies have been associated with a high prevalence of B19V DNA in endothelial cells of the myocardium. To elucidate the mechanisms of B19V uptake into endothelium, we first analyzed the surface expression of the well-characterized primary B19V receptor P antigen and the putative coreceptors α5β1 integrins and Ku80 antigen on primary and permanent endothelial cells. The receptor expression pattern and also the primary attachment levels were similar to those in the UT7/Epo-S1 cell line regarded as functional for B19V entry, but internalization of the virus was strongly reduced. As an alternative B19V uptake mechanism in endothelial cells, we demonstrated antibody-dependent enhancement (ADE), with up to a 4,000-fold increase in B19V uptake in the presence of B19V-specific human antibodies. ADE was mediated almost exclusively at the level of virus internalization, with efficient B19V translocation to the nucleus. In contrast to monocytes, where ADE of B19V has been described previously, enhancement does not rely on interaction of the virus-antibody complexes with Fc receptors (FcRs), but rather, involves an alternative mechanism mediated by the heat-sensitive complement factor C1q and its receptor, CD93. Our results suggest that ADE represents the predominant mechanism of endothelial B19V infection, and it is tempting to speculate that it may play a role in the pathogenicity of cardiac B19V infection. IMPORTANCE Both efficient entry and productive infection of human parvovirus B19 (B19V) seem to be limited to erythroid progenitor cells. However, in vivo, the viral DNA can also be detected in additional cell types, such as endothelial cells of the myocardium, where its presence has been associated with acute and chronic inflammatory cardiomyopathies. In this study, we

  10. Evaluation of Populus and Salix continuously irrigated with landfill leachate I. Genotype-specific elemental phytoremediation.

    PubMed

    Zalesny, Ronald S; Bauer, Edmund O

    2007-01-01

    There is a need for the identification and selection of specific tree genotypes that can sequester elements from contaminated soils, with elevated rates of uptake. We irrigated Populus (DN17, DN182, DN34, NM2, NM6) and Salix (94003, 94012, S287, S566, SX61) genotypes planted in large soil-filled containers with landfill leachate or municipal water and tested for differences in inorganic element concentrations (P, K, Ca, Mg, S, Zn, B, Mn, Fe, Cu, Al, Na, and Cl) in the leaves, stems, and roots. Trees were irrigated with leachate or water during the final 12 wk of the 18-wk study. Genotype-specific uptake existed. For genera, tissue concentrations exhibited four responses. First, Populus had the greatest uptake of P, K, S, Cu, and Cl. Second, Salix exhibited the greatest uptake of Zn, B, Fe, and Al. Third, Salix had greater concentrations of Ca and Mg in leaves, while Populus had greater concentrations in stems and roots. Fourth, Populus had greater concentrations of Mn and Na in leaves and stems, while Salix had greater concentrations in roots. Populus deltoides x P. nigra clones exhibited better overall phytoremediation than the P. nigra x P. maximowiczii genotypes tested. Phytoremediation for S. purpurea clones 94003 and 94012 was generally less than for other Salix genotypes. Overall, concentrations of elements in the leaves, stems, and roots corroborated those in the plant-sciences literature. Uptake was dependent upon the specific genotype for most elements. Our results corroborated the need for further testing and selecting of specific clones for various phytoremediation needs, while providing a baseline for future researchers developing additional studies and resource managers conducting on-site remediation.

  11. Parvovirus B19 Uptake Is a Highly Selective Process Controlled by VP1u, a Novel Determinant of Viral Tropism

    PubMed Central

    Leisi, Remo; Ruprecht, Nico; Kempf, Christoph

    2013-01-01

    The VP1 unique region (VP1u) of human parvovirus B19 (B19V) is the immunodominant part of the viral capsid. Originally inaccessible, the VP1u becomes exposed upon primary attachment to the globoside receptor. To study the function of the exposed VP1u in B19V uptake, we expressed this region as a recombinant protein. Here, we report that purified recombinant VP1u binds and is internalized in UT7/Epo cells. By means of truncations and specific antibodies, we identified the most N-terminal amino acid residues of VP1u as the essential region for binding and internalization. Furthermore, the recombinant VP1u was able to block B19V uptake, suggesting that the protein and the virus undertake the same internalization pathway. Assays with different erythroid and nonerythroid cell lines showed that the N-terminal VP1u binding was restricted to a few cell lines of the erythroid lineage, which were also the only cells that allowed B19V internalization and infection. These results together indicate that the N-terminal region of VP1u is responsible for the internalization of the virus and that the interacting receptor is restricted to B19V-susceptible cells. The highly selective uptake mechanism represents a novel determinant of the tropism and pathogenesis of B19V. PMID:24067971

  12. Parvovirus B19 uptake is a highly selective process controlled by VP1u, a novel determinant of viral tropism.

    PubMed

    Leisi, Remo; Ruprecht, Nico; Kempf, Christoph; Ros, Carlos

    2013-12-01

    The VP1 unique region (VP1u) of human parvovirus B19 (B19V) is the immunodominant part of the viral capsid. Originally inaccessible, the VP1u becomes exposed upon primary attachment to the globoside receptor. To study the function of the exposed VP1u in B19V uptake, we expressed this region as a recombinant protein. Here, we report that purified recombinant VP1u binds and is internalized in UT7/Epo cells. By means of truncations and specific antibodies, we identified the most N-terminal amino acid residues of VP1u as the essential region for binding and internalization. Furthermore, the recombinant VP1u was able to block B19V uptake, suggesting that the protein and the virus undertake the same internalization pathway. Assays with different erythroid and nonerythroid cell lines showed that the N-terminal VP1u binding was restricted to a few cell lines of the erythroid lineage, which were also the only cells that allowed B19V internalization and infection. These results together indicate that the N-terminal region of VP1u is responsible for the internalization of the virus and that the interacting receptor is restricted to B19V-susceptible cells. The highly selective uptake mechanism represents a novel determinant of the tropism and pathogenesis of B19V.

  13. Parvovirus B19 does not bind to membrane-associated globoside in vitro.

    PubMed

    Kaufmann, Bärbel; Baxa, Ulrich; Chipman, Paul R; Rossmann, Michael G; Modrow, Susanne; Seckler, Robert

    2005-02-01

    The glycosphingolipid globoside (globotetraosylceramide, Gb4Cer) has been proposed to be the cellular receptor of human parvovirus B19. Quantitative measurements of the binding of parvovirus B19 to Gb4Cer were performed to explore the molecular basis of the virus tropism. Solid-phase assays with fluorescence-labeled liposomes or 125iodine-labeled empty capsids were used to characterize the specificity of binding. In addition, surface plasmon resonance on lipid layers, as well as isothermal titration microcalorimetry, was utilized for real-time analysis of the virus-receptor interaction. These studies did not confirm binding of Gb4Cer to recombinant B19 VP2 capsids, suggesting that Gb4Cer does not function on its own as the cellular receptor of human parvovirus B19, but might be involved in a more complex recognition event. The biochemical results were further confirmed by cryo-electron microscopy image reconstructions at 10 A resolution, in which the structures of empty capsids were compared with empty capsids incubated with Gb4Cer.

  14. Highly specific and sensitive electrochemical genotyping via gap ligation reaction and surface hybridization detection.

    PubMed

    Huang, Yong; Zhang, Yan-Li; Xu, Xiangmin; Jiang, Jian-Hui; Shen, Guo-Li; Yu, Ru-Qin

    2009-02-25

    This paper developed a novel electrochemical genotyping strategy based on gap ligation reaction with surface hybridization detection. This strategy utilized homogeneous enzymatic reactions to generate molecular beacon-structured allele-specific products that could be cooperatively annealed to capture probes stably immobilized on the surface via disulfide anchors, thus allowing ultrasensitive surface hybridization detection of the allele-specific products through redox tags in close proximity to the electrode. Such a unique biphasic architecture provided a universal methodology for incorporating enzymatic discrimination reactions in electrochemical genotyping with desirable reproducibility, high efficiency and no interferences from interficial steric hindrance. The developed technique was demonstrated to show intrinsic high sensitivity for direct genomic analysis, and excellent specificity with discriminativity of single nucleotide variations.

  15. Linking genotypes database with locus-specific database and genotype–phenotype correlation in phenylketonuria

    PubMed Central

    Wettstein, Sarah; Underhaug, Jarl; Perez, Belen; Marsden, Brian D; Yue, Wyatt W; Martinez, Aurora; Blau, Nenad

    2015-01-01

    The wide range of metabolic phenotypes in phenylketonuria is due to a large number of variants causing variable impairment in phenylalanine hydroxylase function. A total of 834 phenylalanine hydroxylase gene variants from the locus-specific database PAHvdb and genotypes of 4181 phenylketonuria patients from the BIOPKU database were characterized using FoldX, SIFT Blink, Polyphen-2 and SNPs3D algorithms. Obtained data was correlated with residual enzyme activity, patients' phenotype and tetrahydrobiopterin responsiveness. A descriptive analysis of both databases was compiled and an interactive viewer in PAHvdb database was implemented for structure visualization of missense variants. We found a quantitative relationship between phenylalanine hydroxylase protein stability and enzyme activity (rs=0.479), between protein stability and allelic phenotype (rs=−0.458), as well as between enzyme activity and allelic phenotype (rs=0.799). Enzyme stability algorithms (FoldX and SNPs3D), allelic phenotype and enzyme activity were most powerful to predict patients' phenotype and tetrahydrobiopterin response. Phenotype prediction was most accurate in deleterious genotypes (≈100%), followed by homozygous (92.9%), hemizygous (94.8%), and compound heterozygous genotypes (77.9%), while tetrahydrobiopterin response was correctly predicted in 71.0% of all cases. To our knowledge this is the largest study using algorithms for the prediction of patients' phenotype and tetrahydrobiopterin responsiveness in phenylketonuria patients, using data from the locus-specific and genotypes database. PMID:24939588

  16. Placental Cellular Immune Response in Women Infected with Human Parvovirus B19 during Pregnancy

    PubMed Central

    Jordan, Jeanne A.; Huff, Dale; DeLoia, Julie A.

    2001-01-01

    Human parvovirus B19 can cause congenital infection with variable morbidity and mortality in the fetus and neonate. Although much information exists on the B19-specific antibody response in pregnant women, little information is available describing the cell-mediated immune (CMI) response at the maternal-fetal interface. The focus of this study was to characterize the CMI response within placentas from women who seroconverted to B19 during their pregnancies and compare it to controls. Immunohistochemical techniques were used to identify the various immune cells and the inflammatory cytokine present within placental tissue sections. Group 1 consisted of placentas from 25 women whose pregnancies were complicated by B19 infection; 6 women with good outcome (near-term or term delivery), and 19 with poor outcome (spontaneous abortion, nonimmune hydrops fetalis, or fetal death). Group 2 consisted of placentas from 20 women whose pregnancies were complicated with nonimmune hydrops fetalis of known, noninfectious etiology. Group 3 consisted of placentas from eight women whose pregnancies ended in either term delivery or elective abortion. The results of the study revealed a statistically significant increase in the number of CD3-positive T cells present within placentas from group 1 compared to group 2 or 3 (13.3 versus 2 and 1, respectively) (P < 0.001). In addition, the inflammatory cytokine interleukin 2 was detected in every placenta within group 1 but was absent from all placentas evaluated from groups 2 and 3. Together, these findings demonstrate evidence for an inflammation-mediated cellular immune response within placentas from women whose pregnancies are complicated with B19 infection. PMID:11238210

  17. High-speed droplet-allele-specific polymerase chain reaction for genotyping of single nucleotide polymorphisms.

    PubMed

    Matsuda, Kazuyuki; Honda, Takayuki

    2015-01-01

    Single nucleotide alternations such as single nucleotide polymorphisms (SNPs) or single nucleotide mutations are useful genetic markers for molecular diagnosis, prognosis, drug response, and predisposition to diseases. Rapid identification of SNPs or mutations is clinically important, especially for determining drug responses and selection of molecular-targeted therapy. Here, we describe a rapid genotyping assay based on the allele-specific polymerase chain reaction (AS-PCR) by using our droplet-PCR machine (droplet-AS-PCR).

  18. Microarrays for high-throughput genotyping of MICA alleles using allele-specific primer extension.

    PubMed

    Baek, I C; Jang, J-P; Choi, H-B; Choi, E-J; Ko, W-Y; Kim, T-G

    2013-10-01

    The role of major histocompatibility complex (MHC) class I chain-related gene A (MICA), a ligand of NKG2D, has been defined in human diseases by its allele associations with various autoimmune diseases, hematopoietic stem cell transplantation (HSCT) and cancer. This study describes a practical system to develop MICA genotyping by allele-specific primer extension (ASPE) on microarrays. From the results of 20 control primers, strict and reliable cut-off values of more than 30,000 mean fluorescence intensity (MFI) as positive and less than 3000 MFI as negative, were applied to select high-quality specific extension primers. Among 55 allele-specific primers, 44 primers could be initially selected as optimal primer. Through adjusting the length, six primers were improved. The other failed five primers were corrected by refractory modification. MICA genotypes by ASPE on microarrays showed the same results as those by nucleotide sequencing. On the basis of these results, ASPE on microarrays may provide high-throughput genotyping for MICA alleles for population studies, disease-gene associations and HSCT.

  19. Does the chicken genotype 'Géline de Touraine' have specific carcass and meat characteristics?

    PubMed

    Baéza, E; Chartrin, P; Le Bihan-Duval, E; Lessire, M; Besnard, J; Berri, C

    2009-05-01

    The aim of this study was to determine the specific characteristics of carcass and meat from an old French chicken breed, the 'Géline de Touraine' (GT), characterised by a very slow-growing rate and usually slaughtered at 120 days of age. For this purpose, we compared the GT with an experimental crossbreed (EC) exhibiting the same growth rate, and with a 'Label rouge' (LR) genotype usually slaughtered at 84 days of age. A total of 250 males and 250 females per genotype were reared by separating sexes and genotypes. The growth performances were recorded. At 84 days of age, 80 birds per sex and per genotype were slaughtered. The frequency of clawing and pecking injuries on the carcass was noted. We also measured the skin colour and the thickness of wing membrane. The relative percentages of carcass, breast, thigh + drumstick, abdominal fat, testis or ovary to body weight were determined. On breast and thigh muscles the ultimate pH (pHu) and colour were measured. The juice loss after 3 days' storage at +4°C and after cooking at 85°C, and the shear force value of Warner-Bratzler were only measured on breast muscles. At 120 days of age, we repeated the same measurements but only on EC and GT genotypes in order to compare birds at the same age or at the respective slaughter age for each production. Whatever the slaughter age, the body weight of males was always higher than that of the females but the carcass yield was similar for both sexes. The females had higher breast yield and carcass fatness but lower thigh + drumstick yield than the males. The yellowness of skin and meat was higher for the females than for the males while the contrary was observed for the redness of the meat. The breast meat of the females also had higher cooking loss than that of the males. GT and EC birds exhibited a higher occurrence of carcass defects and a higher pHu in meat than LR birds. The GT chickens were characterised by a lower breast yield, a higher fattiness and an earlier sexual

  20. Seroprevalence of parvovirus B19 in the German population

    PubMed Central

    RÖHRER, C.; GÄRTNER, B.; SAUERBREI, A.; BÖHM, S.; HOTTENTRÄGER, B.; RAAB, U.; THIERFELDER, W.; WUTZLER, P.; MODROW, S.

    2008-01-01

    SUMMARY Acute parvovirus B19 infection is a risk for pregnant women. After vertical transmission the infected fetus may develop hydrops fetalis. Since B19 infection occurs mainly during childhood, children represent a main source for virus transmission. In order to determine whether certain groups in the German population show increased risks for B19 infection we analysed the seroprevalence using 6583 sera collected from adults in former Eastern and Western Germany during the German National Health Survey and 649 sera from healthy Thuringian children and adolescents. In adults the overall seroprevalence was 72·1%, rising from 20·4% in children (1–3 years) and 66·9% in adolescents (18–19 years) to 79·1% in the elderly (65–69 years). Significant differences were observed between females (73·3%) and males (70·9%) and between inhabitants of small (74·8%) and big cities (69·0%) but not between people of the former Eastern (72·8%) and Western states (72·0%) of Germany. For women during childbearing age (18–49 years) highest values were observed in those living together with two or more children (81·6%) and in women with occupational contact with children aged <6 years (88·9%). In contrast seroprevalence was significantly lower in age-matched female singles (64·8%) and in women with occupational contact with children aged >6 years and adolescents (63·8%). PMID:18198003

  1. Soybean aphid intrabiotype variability based on colonization of specific soybean genotypes.

    PubMed

    Pawlowski, Michelle; Hill, Curtis B; Voegtlin, David J; Hartman, Glen L

    2015-12-01

    The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is one of the most destructive insect pests on soybeans in the United States. One method for managing this pest is through host plant resistance. Since its arrival in 2000, 4 aphid biotypes have been identified that are able to overcome soybean aphid resistance (Rag) genes. A soybean aphid isolate collected from Moline, Illinois readily colonized soybean plants with the soybean aphid resistance gene Rag2, unlike biotypes 1 and 2, but similar to soybean aphid biotype 3. Two no-choice experiments compared the virulence of the Moline isolate with biotype 3. In both experiments, differences in aphid population counts were not significant (P > 0.05) on soybean genotypes LD08-12957a (Rag2) and LD11-5413a (Rag2), but the aphid counts for the Moline isolate were significantly (P < 0.05) lower than the aphid counts for the biotype 3 isolate on the soybean genotypes Dowling (Rag1), LD05-16611 (Rag1), LD11-4576a (Rag1), and PI 567598B (rag1b and rag3). The Moline isolate was a variant of aphid biotype 3, which is the first report showing that soybean aphid isolates classified as the same biotype, based on virulence against specific Rag genes, can differ in aggressiveness or ability to colonize specific host genotypes.

  2. Identification of Past and Recent Parvovirus B19 Infection in Immunocompetent Individuals by Quantitative PCR and Enzyme Immunoassays: a Dual-Laboratory Study

    PubMed Central

    Hedman, Lea; Dhanilall, Pravesh; Kantola, Kalle; Nurmi, Visa; Söderlund-Venermo, Maria; Brown, Kevin E.; Hedman, Klaus

    2014-01-01

    Parvovirus B19 (B19V) is a member of the family Parvoviridae, genus Erythrovirus. B19V-specific IgG and IgM react differently against conformational and linear epitopes of VP1 and VP2 antigens, leading to the development of IgG avidity and epitope type specificity (ETS) enzyme immunoassays (EIAs) for distinguishing past from recent infection. Additionally, B19V viral load determination (by quantitative PCR [qPCR]) is increasingly used in the staging of B19V infection. In this study, the utility of these methods is compared. A panel of 78 sera was jointly tested by the Virus Reference Department (VRD), London, United Kingdom, and the Haartman Institute (HI), Helsinki, Finland, using a number of EIAs, e.g., B19V-specific IgG and IgM, IgG avidity, and ETS EIAs. At VRD, the sera were also tested by a B19V viral load PCR (qPCR). By consensus analysis, 43 (55.1%) sera represented past infection, 28 (35.9%) sera represented recent infection, and 7 (9.0%) sera were indeterminate. Both VRD B19V qPCR and HI B19V VP2 IgM EIA gave the highest agreement with consensus interpretation for past or recent infection, with an overall agreement of 99% (95% confidence interval [CI], 92 to 100) and positive predictive value (PPV) of 100% (95% CI, 87 to 100). Nine sera designated as representing past infection by consensus analysis were B19V IgM positive by a commercial VRD B19V IgM EIA and B19V IgM negative by a new HI in-house B19V VP2 IgM EIA. A new VRD B19V IgG avidity EIA showed good (>95%) agreement (excluding equivocal results) with consensus interpretations for past or recent infection. Correct discrimination of past from recent B19V infection was achieved through application of qPCR or by appropriate selection of EIAs. PMID:24403307

  3. Human Papillomavirus Genotype-Specific Prevalence Across the Continuum of Cervical Neoplasia and Cancer

    PubMed Central

    Joste, Nancy E.; Ronnett, Brigitte M.; Hunt, William C.; Pearse, Amanda; Langsfeld, Erika; Leete, Thomas; Jaramillo, MaryAnn; Stoler, Mark H.; Castle, Philip E.; Wheeler, Cosette M.

    2014-01-01

    Background The New Mexico HPV Pap Registry was established to measure the impact of cervical cancer prevention strategies in the United States. Prior to widespread HPV vaccine implementation, we established the baseline prevalence for a broad spectrum of HPV genotypes across the continuum of cervical intraepithelial neoplasia (CIN) and cancer. Methods A population-based sample of 6,272 tissue specimens were tested for 37 HPV genotypes. The number of specimens tested within each diagnostic category was: 541 negative, 1,411 CIN grade 1 (CIN1), 2,226 CIN grade 2 (CIN2), and 2,094 CIN grade 3 (CIN3) or greater. Age-specific HPV prevalence was estimated within categories for HPV genotypes targeted by HPV vaccines. Results The combined prevalence of HPV genotypes included in the quadrivalent and nonavalent vaccines increased from 15.3% and 29.3% in CIN1 to 58.4% and 83.7% in CIN3, respectively. The prevalence of HPV types included in both vaccines tended to decrease with increasing age for CIN1, CIN2, CIN3, and squamous cell carcinoma, most notably for CIN3 and SCC. The six most common HPV types in descending order of prevalence were HPV-16, −31, −52, −58, −33, and −39 for CIN3 and HPV-16, −18, −31, −45, −52, and −33 for invasive cancers. Conclusions Health economic modeling of HPV vaccine impact should consider age-specific differences in HPV prevalence. Impact Population-based HPV prevalence in CIN is not well described but is requisite for longitudinal assessment of vaccine impact and to understand the effectiveness and performance of various cervical screening strategies in vaccinated and unvaccinated women. PMID:25363635

  4. Resolution of Ambiguous HLA Genotyping in Korean by Multi-Group-Specific Sequence-Based Typing

    PubMed Central

    Park, Yongjung; Yoon, Cha Eun; Kwon, Oh-Joong; Kim, Yu-Seun

    2014-01-01

    Purpose To evaluate a multi-group-specific sequence-based typing (SBT) method for resolving ambiguous results from human leukocyte antigen (HLA) genotyping. Materials and Methods A total of 50 samples that showed ambiguous genotypes for at least two HLA loci from HLA-A, -B, -C and -DRB1 by the conventional SBT assay were evaluated using a new SBT test, the AVITA plus assay. The most likely HLA genotypes for the respective samples considering allele frequencies in Korean were concordant between the AVITA and conventional SBT assays. Results An average of 3.3 loci among the HLA-A, -B, -C and -DRB1 loci per sample gave results with two or more possible allele combinations with the conventional SBT, and 48 (96.0%) out of 50 showed reduced numbers of possible genotypes for at least one HLA locus with the AVITA. A total of 41, 43, 42, and 38 cases among the 50 samples showed ambiguous results for HLA-A, -B, -C, and -DRB1 typing by the conventional SBT, respectively. The average numbers of possible allele combinations for the respective four HLA loci were 8.2, 6.7, 5.9, and 3.2, and they were reduced to 1.5, 2.2, 4.4, and 1.8, respectively, by the AVITA. Ambiguity was resolved by the AVITA in 33 (80.5%), 31 (72.1%), 17 (40.5%) and 28 (73.7%) samples among the ambiguous cases from the conventional SBT for HLA-A, -B, -C, and -DRB1 typing, respectively. Conclusion The multi-group-specific SBT method considerably reduced the number of ambiguous results, and thus may be useful for accurate HLA typing in clinical laboratories. PMID:24954331

  5. Association of severe malaria with a specific Plasmodium falciparum genotype in French Guiana.

    PubMed

    Ariey, F; Hommel, D; Le Scanf, C; Duchemin, J B; Peneau, C; Hulin, A; Sarthou, J L; Reynes, J M; Fandeur, T; Mercereau-Puijalon, O

    2001-07-15

    Why severe Plasmodium falciparum malaria occurs in only a small percentage of patients is unclear. The possibility that specific parasite characteristics contribute to severity has been investigated in French Guiana, a hypoendemic area, where parasite diversity is low and all patients with severe cases are referred to a single intensive care unit. Parasite genotyping in geographically and temporally matched patients with mild and severe disease showed that the association of a specific msp-1 allele (B-K1) with a specific var gene (var-D) was overrepresented among patients with severe versus mild disease (47% vs. 3%, respectively; P<.001). Moreover, this genotype combination was consistently observed in the most severe clinical cases. Reverse-transcription polymerase chain reaction demonstrated programmed expression of var-D in vivo, which is consistent with its potential implication in severe disease. These results provide field evidence of an association of severe malaria with specific genetic characteristics of parasites and open the way for intervention strategies targeting key virulence factors of parasites.

  6. High prevalence and two dominant host-specific genotypes of Coxiella burnetii in U.S. milk

    PubMed Central

    2014-01-01

    Background Coxiella burnetii causes Q fever in humans and Coxiellosis in animals; symptoms range from general malaise to fever, pneumonia, endocarditis and death. Livestock are a significant source of human infection as they shed C. burnetii cells in birth tissues, milk, urine and feces. Although prevalence of C. burnetii is high, few Q fever cases are reported in the U.S. and we have a limited understanding of their connectedness due to difficulties in genotyping. Here, we develop canonical SNP genotyping assays to evaluate spatial and temporal relationships among C. burnetii environmental samples and compare them across studies. Given the genotypic diversity of historical collections, we hypothesized that the current enzootic of Coxiellosis is caused by multiple circulating genotypes. We collected A) 23 milk samples from a single bovine herd, B) 134 commercial bovine and caprine milk samples from across the U.S., and C) 400 bovine and caprine samples from six milk processing plants over three years. Results We detected C. burnetii DNA in 96% of samples with no variance over time. We genotyped 88.5% of positive samples; bovine milk contained only a single genotype (ST20) and caprine milk was dominated by a second type (mostly ST8). Conclusions The high prevalence and lack of genotypic diversity is consistent with a model of rapid spread and persistence. The segregation of genotypes between host species is indicative of species-specific adaptations or dissemination barriers and may offer insights into the relative lack of human cases and characterizing genotypes. PMID:24533573

  7. Study of chronic hemolytic anaemia patients in Rio de Janeiro: prevalence of anti-human parvovirus B19 IgG antibodies and the development aplastic crises.

    PubMed

    Sant'Anna, Anadayr L M; Garcia, Rita de Cássia N Cubel; Marzoche, Mônica; da Rocha, Heloisa Helena A Gallo; Paula, Maria Tereza M; Lobo, Clarisse C; Nascimento, Jussara P

    2002-01-01

    The prevalence of anti-human parvovirus B19 IgG antibodies was determined in sera from 165 chronic hemolytic anemia patients, receiving medical care at Instituto Estadual de Hematologia (IEHE), Rio de Janeiro, during the year of 1994. This sample represents around 10% of the chronic hemolytic anemia patients attending at IEHE. Most of these patients (140) have sickle cell disease. Anti-B19 IgG antibodies were detected in 32.1% of patients. No statistically significant difference (p > 0.05) was seen between IgG antibody prevalence in male (27.8%) and female (35.5%) patients. Anti-B19 IgG antibodies were more frequent in older (37.6%) than younger (28.2%) than 20 years old patients, although this difference had no statistical significance (p > 0.05). Anti-B19 IgG antibody prevalence showed that 67.9% of patients enrolled in the study were susceptible to B19 acute infection. With the aim to detect acute B19 infection, patients follow up continued until February 1996. During this period four patients presented transient aplastic crisis due to human parvovirus B19 as confirmed by the detection of specific IgM antibodies. All four patients were younger than 20 years old, and 3 were younger than 10 years old. Three of them were sickle cell disease patients. Three of the four acute B19 infection occurred during 1994 springtime.

  8. Apolipoprotein E and Alzheimer disease: Genotype-specific risks by age and sex

    SciTech Connect

    Bickeboeller, H. |; Babron, M.C.; Clerget-Darpoux, F.

    1997-02-01

    The distribution of apolipoprotein E (APOE) genotypes as a function of age and sex has been examined in a French population of 417 Alzheimer disease (AD) patients and 1,030 control subjects. When compared to the APOE {epsilon}3 allele, an increased risk associated with the APOE {epsilon}4 allele (odds ratio [OR] [{epsilon}4] = 2.7 with 95% confidence interval [CI] = 2.0-3.6; P < .001) and a protective effect of the APOE {epsilon}2 allele (OR[{epsilon}2] = 0.5 with 95% CI = 0.3-0.98; P = .012) were retrieved. An effect of the {epsilon}4 allele dosage on susceptibility was confirmed (OR[{epsilon}4/{epsilon}4] vs. the {epsilon}3/{epsilon}3 genotype = 11.2 [95% CI = 4.0-31.6]; OR[{epsilon}3/{epsilon}4] vs. the {epsilon}3/{epsilon}3 genotype = 2.2 [95% Cl = 1.5-3.5]). The frequency of the {epsilon}4 allele was lower in male cases than in female cases, but, since a similar difference was found in controls, this does not lead to a difference in OR between sex. ORs for the {epsilon}4 allele versus the {epsilon}3 allele, OR({epsilon}4), were not equal in all age classes: OR({epsilon}4) in the extreme groups with onset at < 60 years or > 79 years were significantly lower than those from the age groups 60-79 years. In {epsilon}3/{epsilon}4 individuals, sex-specific lifetime risk estimates by age 85 years (i.e., sex-specific penetrances by age 85 years) were 0.14 (95% CI 0.04-0.30) for men and 0.17 (95% CI 0.09-0.28) for women. 53 refs., 1 fig., 3 tabs.

  9. Direct and correlated responses to selection in a host-parasite system: testing for the emergence of genotype specificity.

    PubMed

    Nidelet, Thibault; Kaltz, Oliver

    2007-08-01

    Genotype x environment interactions can facilitate coexistence of locally adapted specialists. Interactions evolve if adaptation to one environment trades off with performance in others. We investigated whether evolution on one host genotype traded off with performance on others in long-term experimental populations of different genotypes of the protozoan Paramecium caudatum, infected with the bacterial parasite Holospora undulata. A total of nine parasite selection lines evolving on three host genotypes and the ancestral parasite were tested in a cross-infection experiment. We found that evolved parasites produced more infections than did the ancestral parasites, both on host genotypes they had evolved on (positive direct response to selection) and on genotypes they had not evolved on (positive correlated response to selection). On two host genotypes, a negative relationship between direct and correlated responses indicated pleiotropic costs of adaptation. On the third, a positive relationship suggested cost-free adaptation. Nonetheless, on all three hosts, resident parasites tended to be superior to the average nonresident parasite. Thus genotype specificity (i.e., patterns of local adaptation) may evolve without costs of adaptation, as long as direct responses to selection exceed correlated responses.

  10. Plant genotype-specific archaeal and bacterial endophytes but similar Bacillus antagonists colonize Mediterranean olive trees

    PubMed Central

    Müller, Henry; Berg, Christian; Landa, Blanca B.; Auerbach, Anna; Moissl-Eichinger, Christine; Berg, Gabriele

    2015-01-01

    Endophytes have an intimate and often symbiotic interaction with their hosts. Less is known about the composition and function of endophytes in trees. In order to evaluate our hypothesis that plant genotype and origin have a strong impact on both, endophytes of leaves from 10 Olea europaea L. cultivars from the Mediterranean basin growing at a single agricultural site in Spain and from nine wild olive trees located in natural habitats in Greece, Cyprus, and on Madeira Island were studied. The composition of the bacterial endophytic communities as revealed by 16S rRNA gene amplicon sequencing and the subsequent PCoA analysis showed a strong correlation to the plant genotypes. The bacterial distribution patterns were congruent with the plant origins in “Eastern” and “Western” areas of the Mediterranean basin. Subsequently, the endophytic microbiome of wild olives was shown to be closely related to those of cultivated olives of the corresponding geographic origins. The olive leaf endosphere harbored mostly Proteobacteria, followed by Firmicutes, Actinobacteria, and Bacteroidetes. The detection of a high portion of archaeal taxa belonging to the phyla Thaumarchaeota, Crenarchaeota, and Euryarchaeota in the amplicon libraries was an unexpected discovery, which was confirmed by quantitative real-time PCR revealing an archaeal portion of up to 35.8%. Although the function of these Archaea for their host plant remains speculative, this finding suggests a significant relevance of archaeal endophytes for plant–microbe interactions. In addition, the antagonistic potential of culturable endophytes was determined; all isolates with antagonistic activity against the olive-pathogenic fungus Verticillium dahliae Kleb. belong to Bacillus amyloliquefaciens. In contrast to the specific global structural diversity, BOX-fingerprints of the antagonistic Bacillus isolates were highly similar and independent of the olive genotype from which they were isolated. PMID:25784898

  11. Plant genotype-specific archaeal and bacterial endophytes but similar Bacillus antagonists colonize Mediterranean olive trees.

    PubMed

    Müller, Henry; Berg, Christian; Landa, Blanca B; Auerbach, Anna; Moissl-Eichinger, Christine; Berg, Gabriele

    2015-01-01

    Endophytes have an intimate and often symbiotic interaction with their hosts. Less is known about the composition and function of endophytes in trees. In order to evaluate our hypothesis that plant genotype and origin have a strong impact on both, endophytes of leaves from 10 Olea europaea L. cultivars from the Mediterranean basin growing at a single agricultural site in Spain and from nine wild olive trees located in natural habitats in Greece, Cyprus, and on Madeira Island were studied. The composition of the bacterial endophytic communities as revealed by 16S rRNA gene amplicon sequencing and the subsequent PCoA analysis showed a strong correlation to the plant genotypes. The bacterial distribution patterns were congruent with the plant origins in "Eastern" and "Western" areas of the Mediterranean basin. Subsequently, the endophytic microbiome of wild olives was shown to be closely related to those of cultivated olives of the corresponding geographic origins. The olive leaf endosphere harbored mostly Proteobacteria, followed by Firmicutes, Actinobacteria, and Bacteroidetes. The detection of a high portion of archaeal taxa belonging to the phyla Thaumarchaeota, Crenarchaeota, and Euryarchaeota in the amplicon libraries was an unexpected discovery, which was confirmed by quantitative real-time PCR revealing an archaeal portion of up to 35.8%. Although the function of these Archaea for their host plant remains speculative, this finding suggests a significant relevance of archaeal endophytes for plant-microbe interactions. In addition, the antagonistic potential of culturable endophytes was determined; all isolates with antagonistic activity against the olive-pathogenic fungus Verticillium dahliae Kleb. belong to Bacillus amyloliquefaciens. In contrast to the specific global structural diversity, BOX-fingerprints of the antagonistic Bacillus isolates were highly similar and independent of the olive genotype from which they were isolated. PMID:25784898

  12. Infection of the erythroid cell line, KU812Ep6 with human parvovirus B19 and its application to titration of B19 infectivity.

    PubMed

    Miyagawa, E; Yoshida, T; Takahashi, H; Yamaguchi, K; Nagano, T; Kiriyama, Y; Okochi, K; Sato, H

    1999-12-01

    A human parvovirus B19 (B19) infectivity assay was developed using the erythroid cell line, KU812Ep6. KU812Ep6 was cloned for high efficiency infection with B19 in vitro, in the presence of erythropoietin by a limiting dilution method from the parent cell line, KU812. B19 was effectively propagated in KU812Ep6 and was detected for B19 antigens, VP1 and VP2. The titers of B19 positive sera measured with KU812Ep6 cells were in the range of 10(6) to 10(8) TCID50 ml. This KU812Ep6 infectivity assay had a 10(3)-10(4.5) higher sensitivity than the colony forming unit-erythroid (CFU-e) injury assay. It was calculated that one TCID50 needed 10(3) B19 genome copies, judging from the infectivity assay and semi-quantitative PCR. The KU812Ep6 infectivity assay was also used to determine infectivity of B19 in vitro, and to evaluate inactivation, as well as clearance of the virus. The inactivation of B19 by heating was carried out and infectivity declined from 10(4) TCID50 ml to < 10 TCID50 ml (lower limit of detection) at 60 degrees C for 3 h or at 70 degrees C for 30 min, but only decreased to 10(2.5) TCID50 ml at 50 degrees C for 8 h.

  13. 76 FR 15823 - Airworthiness Directives; Bombardier, Inc. Model CL-600-2B19 (Regional Jet Series 100 & 440...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-22

    ... published in the Federal Register on August 5, 2010 (75 FR 47249). That NPRM proposed to correct an unsafe... incorporated in production, and therefore are not affected by the identified unsafe condition. Specifically, Model CL-600-2B19 (Regional Jet Series 100 & 440) airplanes, serial numbers 8108 through 8111 have...

  14. Manganese Deficiency Leads to Genotype-Specific Changes in Fluorescence Induction Kinetics and State Transitions1[C][OA

    PubMed Central

    Husted, Søren; Laursen, Kristian H.; Hebbern, Christopher A.; Schmidt, Sidsel B.; Pedas, Pai; Haldrup, Anna; Jensen, Poul E.

    2009-01-01

    Barley (Hordeum vulgare) genotypes display a marked difference in their ability to tolerate growth at low manganese (Mn) concentrations, a phenomenon designated as differential Mn efficiency. Induction of Mn deficiency in two genotypes differing in Mn efficiency led to a decline in the quantum yield efficiency for both, although faster in the Mn-inefficient genotype. Leaf tissue and thylakoid Mn concentrations were reduced under Mn deficiency, but no difference between genotypes was observed and no visual Mn deficiency symptoms were developed. Analysis of the fluorescence induction kinetics revealed that in addition to the usual O-J-I-P steps, clear K and D steps were developed in the Mn-inefficient genotype under Mn deficiency. These marked changes indicated damages to photosystem II (PSII). This was further substantiated by state transition measurements, indicating that the ability of plants to redistribute excitation energy was reduced. The percentage change in state transitions for control plants with normal Mn supply of both genotypes was 9% to 11%. However, in Mn-deficient leaves of the Mn-inefficient genotypes, state transitions were reduced to less than 1%, whereas no change was observed for the Mn-efficient genotypes. Immunoblotting and the chlorophyll a/b ratio confirmed that Mn deficiency in general resulted in a significant reduction in abundance of PSII reaction centers relative to the peripheral antenna. In addition, PSII appeared to be significantly more affected by Mn limitation than PSI. However, the striking genotypic differences observed in Mn-deficient plants, when analyzing state transitions and fluorescence induction kinetics, could not be correlated with specific changes in photosystem proteins. Thus, there is no simple linkage between protein expression and the differential reduction in state transition and fluorescence induction kinetics observed for the genotypes under Mn deficiency. PMID:19369593

  15. Genotype-specific regulation of oral innate immunity by T2R38 taste receptor.

    PubMed

    Gil, Sucheol; Coldwell, Susan; Drury, Jeanie L; Arroyo, Fabiola; Phi, Tran; Saadat, Sanaz; Kwong, Danny; Chung, Whasun Oh

    2015-12-01

    The bitter taste receptor T2R38 has been shown to regulate mucosal innate immune responses in the upper airway epithelium. Furthermore, SNPs in T2R38 influence the sensitivity to 6-n-propylthiouracil (PROP) and are associated with caries risk/protection. However, no study has been reported on the role of T2R38 in the innate immune responses to oral bacteria. We hypothesize that T2R38 regulates oral innate immunity and that this regulation is genotype-specific. Primary gingival epithelial cells carrying three common genotypes, PAV/PAV (PROP super-taster), AVI/PAV (intermediate) and AVI/AVI (non-taster) were stimulated with cariogenic bacteria Streptococcus mutans, periodontal pathogen Porphyromonas gingivalis or non-pathogen Fusobacterium nucleatum. QRT-PCR analyzed T2R38 mRNA, and T2R38-specific siRNA and ELISA were utilized to evaluate induction of hBD-2 (antimicrobial peptide), IL-1α and IL-8 in various donor-lines. Experiments were set up in duplicate and repeated three times. T2R38 mRNA induction in response to S. mutans was highest in PAV/PAV (4.3-fold above the unstimulated controls; p<0.05), while lowest in AVI/AVI (1.2-fold). In PAV/PAV, hBD-2 secretion in response to S. mutans was decreased by 77% when T2R38 was silenced. IL-1α secretion was higher in PAV/PAV compared to AVI/PAV or AVI/AVI with S. mutans stimulation, but it was reduced by half when T2R38 was silenced (p<0.05). In response to P. gingivalis, AVI/AVI showed 4.4-fold increase (p<0.05) in T2R38 expression, whereas the levels in PAV/PAV and AVI/PAV remained close to that of the controls. Secretion levels of IL-1α and IL-8 decreased in AVI/AVI in response to P. gingivalis when T2R38 was silenced (p<0.05), while the changes were not significant in PAV/PAV. Our data suggest that the regulation of gingival innate immunity by T2R38 is genotype-dependent and that the ability to induce a high level of hBD-2 by PAV/PAV carriers may be a reason for protection against caries in this group. PMID

  16. Cynomolgus monkeys (Macaca fascicularis) experimentally infected with B19V and hepatitis A virus: no evidence of the co-infection as a cause of acute liver failure

    PubMed Central

    Leon, Luciane Almeida Amado; Marchevsky, Renato Sergio; Gaspar, Ana Maria Coimbra; Garcia, Rita de Cassia Nasser Cubel; de Almeida, Adilson José; Pelajo-Machado, Marcelo; de Castro, Tatiana Xavier; do Nascimento, Jussara Pereira; Brown, Kevin E; Pinto, Marcelo Alves

    2016-01-01

    This study was conducted to analyse the course and the outcome of the liver disease in the co-infected animals in order to evaluate a possible synergic effect of human parvovirus B19 (B19V) and hepatitis A virus (HAV) co-infection. Nine adult cynomolgus monkeys were inoculated with serum obtained from a fatal case of B19V infection and/or a faecal suspension of acute HAV. The presence of specific antibodies to HAV and B19V, liver enzyme levels, viraemia, haematological changes, and necroinflammatory liver lesions were used for monitoring the infections. Seroconversion was confirmed in all infected groups. A similar pattern of B19V infection to human disease was observed, which was characterised by high and persistent viraemia in association with reticulocytopenia and mild to moderate anaemia during the period of investigation (59 days). Additionally, the intranuclear inclusion bodies were observed in pro-erythroblast cell from an infected cynomolgus and B19V Ag in hepatocytes. The erythroid hypoplasia and decrease in lymphocyte counts were more evident in the co-infected group. The present results demonstrated, for the first time, the susceptibility of cynomolgus to B19V infection, but it did not show a worsening of liver histopathology in the co-infected group. PMID:27074255

  17. Glycan Specificity of P[19] Rotavirus and Comparison with Those of Related P Genotypes

    PubMed Central

    Liu, Yang; Ramelot, Theresa A.; Huang, Pengwei; Liu, Yan; Li, Zhen; Feizi, Ten; Zhong, Weiming; Wu, Fang-Tzy; Tan, Ming; Kennedy, Michael A.

    2016-01-01

    over other P genotypes. Elucidation of the molecular bases for strain-specific host ranges and cross-species transmission of these human and animal RVs is important to understand RV epidemiology and disease burden, which may impact development of control and prevention strategies against RV gastroenteritis. PMID:27558427

  18. The role of parvovirus B19 in the pathogenesis of autoimmunity and autoimmune disease.

    PubMed

    Kerr, Jonathan R

    2016-04-01

    Human parvovirus B19 is a single-stranded DNA virus which preferentially targets the erythroblasts in the bone marrow. B19 infection commonly causes erythema infectiosum, arthralgia, fetal death, transient aplastic crisis in patients with shortened red cell survival, and persistent infection in people who are immunocompromised. Less common clinical manifestations include atypical skin rashes, neurological syndromes, cardiac syndromes, and various cytopenias. B19 infection has also been associated with development of a variety of different autoimmune diseases, including rheumatological, neurological, neuromuscular, cardiovascular, haematological, nephrological and metabolic. Production of a variety of autoantibodies has been demonstrated to occur during B19 infection and these have been shown to be key to the pathogenesis of the particular disease process in a significant number of cases, for example, production of rheumatoid factor in cases of B19-associated rheumatoid arthritis and production of anti-glutamic acid decarboxylase (GAD) in patients with B19-associated type 1 diabetes mellitus. B19 infection has also been associated with the development of multiple autoimmune diseases in 12 individuals. Documented mechanisms in B19-associated autoimmunity include molecular mimicry (IgG antibody to B19 proteins has been shown to cross react with a variety of recognised human autoantigens, including collagen II, keratin, angiotensin II type 1 receptor, myelin basic protein, cardiolipin, and platelet membrane glycoprotein IIb/IIIa), B19-induced apoptosis with presentation of self-antigens to T lymphocytes, and the phospholipase activity of the B19 unique VP1 protein. PMID:26644521

  19. Detection of genotype-specific Ehrlichia canis exposure in Brazilian dogs by TRP36 peptide ELISA.

    PubMed

    Aguiar, Daniel M; Zhang, Xiaofeng; Braga, Isis A; Taques, Isis I G G; McBride, Jere W

    2016-02-01

    We recently characterized a novel genotype of Ehrlichia canis based on the tandem repeat (TR) sequence of the TRP36 gene in Brazil. The TR amino acid sequence of the Brazilian (Br) genotype (ASVVPEAE) was divergent from the previously described US genotype (TEDSVSAPA) of E. canis. In this study, we developed an ELISA based on TRP36 TR synthetic peptides from both Br and US E. canis TRP36 genotypes to serologically detect and distinguish infections caused by these genotypes. Sera from 30 Brazilian dogs naturally infected with E. canis, sera from dogs experimentally infected E. canis (Jake and Cuiabá #1 strains) and E. chaffeensis (Arkansas strain) and 12 seronegative E. canis dogs were evaluated. Fifteen naturally infected Brazilian dogs had antibodies that reacted with the US TRP36 (n=9) or Br TRP36 (n=6) only, and 13 dogs had antibodies that reacted with both TPR36 peptides suggesting that these dogs were exposed to both genotypes. Most dogs (n=28) had antibodies that reacted with the highly conserved E. canis TRP19 peptide; however, two dogs had antibodies to E. canis TRP19, but did not have TRP36 antibodies, raising the possibility that another novel TRP36 genotype is circulating in Brazil. Our results demonstrate that synthetic peptides based on the TR region of E. canis TRP36 can be used to serologically distinguish infections or identify coinfections by different genotypes, and to determine the seroprevalence of various E. canis genotypes in Brazil.

  20. Detection of genotype-specific Ehrlichia canis exposure in Brazilian dogs by TRP36 peptide ELISA.

    PubMed

    Aguiar, Daniel M; Zhang, Xiaofeng; Braga, Isis A; Taques, Isis I G G; McBride, Jere W

    2016-02-01

    We recently characterized a novel genotype of Ehrlichia canis based on the tandem repeat (TR) sequence of the TRP36 gene in Brazil. The TR amino acid sequence of the Brazilian (Br) genotype (ASVVPEAE) was divergent from the previously described US genotype (TEDSVSAPA) of E. canis. In this study, we developed an ELISA based on TRP36 TR synthetic peptides from both Br and US E. canis TRP36 genotypes to serologically detect and distinguish infections caused by these genotypes. Sera from 30 Brazilian dogs naturally infected with E. canis, sera from dogs experimentally infected E. canis (Jake and Cuiabá #1 strains) and E. chaffeensis (Arkansas strain) and 12 seronegative E. canis dogs were evaluated. Fifteen naturally infected Brazilian dogs had antibodies that reacted with the US TRP36 (n=9) or Br TRP36 (n=6) only, and 13 dogs had antibodies that reacted with both TPR36 peptides suggesting that these dogs were exposed to both genotypes. Most dogs (n=28) had antibodies that reacted with the highly conserved E. canis TRP19 peptide; however, two dogs had antibodies to E. canis TRP19, but did not have TRP36 antibodies, raising the possibility that another novel TRP36 genotype is circulating in Brazil. Our results demonstrate that synthetic peptides based on the TR region of E. canis TRP36 can be used to serologically distinguish infections or identify coinfections by different genotypes, and to determine the seroprevalence of various E. canis genotypes in Brazil. PMID:26482949

  1. Hepatitis B Virus Genotype Distribution and Genotype-Specific BCP/preCore Substitutions in Acute and Chronic Infections in Argentina

    PubMed Central

    González López Ledesma, María Mora; Mojsiejczuk, Laura Noelia; Rodrigo, Belén; Sevic, Ina; Mammana, Lilia; Galdame, Omar; Gadano, Adrian; Fainboim, Hugo; Campos, Rodolfo; Flichman, Diego

    2015-01-01

    Aim In order to assess Hepatitis B Virus genotype (g) and subgenotype (sg) implications in the course of infection, 234 HBsAg positive patients in different infection stages were characterized (66 acute infections, 63 HBeAg positive chronic infections and 105 anti-HBe positive chronic infections). Results Overall, sgA2 (17.9%), gD (20.9%), sgF1b (34.2%) and sgF4 (19.7%) were the most prevalent. Subgenotype F1b was overrepresented in acute and chronic HBeAg infections (56.1%), whereas gD was the most frequent (40.0%) in anti-HBe positive chronic infections. Among chronic infections, HBeAg positivity rates were 50.0, 12.5, 62.8 and 35.3% for sgA2, gD, sgF1b and sgF4, respectively (p <0.05). A bias toward BCP/preCore mutations was observed among genotypes. In anti-HBe positive chronic infections, sgF1b was more prone to have A1762T/G1764A mutation than sgA2, sgF4 and gD (75.0, 40.0, 33.3 and 31.8%, p<0.005), whereas in the pC region, gD and sgF4 were more likely to have G1896A than sgA2 and sgF1b (81.0, 72.7, 0.0 and 31.3%, p <0.001). The unexpected low frequency of the G1896A mutation in the sgF1b (despite carrying 1858T) prompted us to perform a further analysis in order to identify genotype-specific features that could justify the pattern mutations observed. A region encompassing nucleotides 1720 to 1920 showed the higher dissimilarity between sgF1b and sgF4. Genotypes and subgenotypes carrying the 1727G, 1740C and 1773T polymorphisms were prevented to mutate position 1896. Discussion HBeAg seroconversion is a critical event in the natural history of HBV infection. Differences in the HBeAg positivity rate might be relevant since different studies have observed that delayed HBeAg seroconversion is associated with a more severe clinical course of infection, highlighting the critical role that genotypes/subgenotypes might play in the progression of HBV infection. Polymorphisms in the regions 1720 to 1920 could be involved in the molecular mechanisms underlying

  2. Transcriptome analyses reveal genotype- and developmental stage-specific molecular responses to drought and salinity stresses in chickpea.

    PubMed

    Garg, Rohini; Shankar, Rama; Thakkar, Bijal; Kudapa, Himabindu; Krishnamurthy, Lakshmanan; Mantri, Nitin; Varshney, Rajeev K; Bhatia, Sabhyata; Jain, Mukesh

    2016-01-01

    Drought and salinity are the major factors that limit chickpea production worldwide. We performed whole transcriptome analyses of chickpea genotypes to investigate the molecular basis of drought and salinity stress response/adaptation. Phenotypic analyses confirmed the contrasting responses of the chickpea genotypes to drought or salinity stress. RNA-seq of the roots of drought and salinity related genotypes was carried out under control and stress conditions at vegetative and/or reproductive stages. Comparative analysis of the transcriptomes revealed divergent gene expression in the chickpea genotypes at different developmental stages. We identified a total of 4954 and 5545 genes exclusively regulated in drought-tolerant and salinity-tolerant genotypes, respectively. A significant fraction (~47%) of the transcription factor encoding genes showed differential expression under stress. The key enzymes involved in metabolic pathways, such as carbohydrate metabolism, photosynthesis, lipid metabolism, generation of precursor metabolites/energy, protein modification, redox homeostasis and cell wall component biogenesis, were affected by drought and/or salinity stresses. Interestingly, transcript isoforms showed expression specificity across the chickpea genotypes and/or developmental stages as illustrated by the AP2-EREBP family members. Our findings provide insights into the transcriptome dynamics and components of regulatory network associated with drought and salinity stress responses in chickpea. PMID:26759178

  3. Transcriptome analyses reveal genotype- and developmental stage-specific molecular responses to drought and salinity stresses in chickpea

    PubMed Central

    Garg, Rohini; Shankar, Rama; Thakkar, Bijal; Kudapa, Himabindu; Krishnamurthy, Lakshmanan; Mantri, Nitin; Varshney, Rajeev K.; Bhatia, Sabhyata; Jain, Mukesh

    2016-01-01

    Drought and salinity are the major factors that limit chickpea production worldwide. We performed whole transcriptome analyses of chickpea genotypes to investigate the molecular basis of drought and salinity stress response/adaptation. Phenotypic analyses confirmed the contrasting responses of the chickpea genotypes to drought or salinity stress. RNA-seq of the roots of drought and salinity related genotypes was carried out under control and stress conditions at vegetative and/or reproductive stages. Comparative analysis of the transcriptomes revealed divergent gene expression in the chickpea genotypes at different developmental stages. We identified a total of 4954 and 5545 genes exclusively regulated in drought-tolerant and salinity-tolerant genotypes, respectively. A significant fraction (~47%) of the transcription factor encoding genes showed differential expression under stress. The key enzymes involved in metabolic pathways, such as carbohydrate metabolism, photosynthesis, lipid metabolism, generation of precursor metabolites/energy, protein modification, redox homeostasis and cell wall component biogenesis, were affected by drought and/or salinity stresses. Interestingly, transcript isoforms showed expression specificity across the chickpea genotypes and/or developmental stages as illustrated by the AP2-EREBP family members. Our findings provide insights into the transcriptome dynamics and components of regulatory network associated with drought and salinity stress responses in chickpea. PMID:26759178

  4. Acute parvovirus B19 infection in identical twins unmasking previously unidentified hereditary spherocytosis.

    PubMed

    Forde, Donall G; Cope, Alison; Stone, Ben

    2014-01-01

    Identical Caucasian male twins, previously fit, presented 1 week apart with short histories of fever and lethargy. The twins were febrile at presentation with profound pancytopaenia and evidence of haemolysis. There was no rash or arthralgia. Both required multiple red cell transfusions. The twins had positive IgM serology for Epstein-Barr virus (EBV), cytomegalovirus (CMV) and parvovirus B19. EBV viral capsid antigen and Epstein-Barr nuclear antigen IgGs were also positive however, suggesting past EBV exposure. Parvovirus B19 DNA was detected from peripheral blood PCR; CMV and EBV DNA PCRs were negative. Convalescent serology demonstrated no evolution of the CMV serological response, that is no IgG to CMV developed which implies an initial non-specific polyclonal IgM response. The twins recovered fully over 7 days, the first with a course of prednisolone and the second spontaneously. They were diagnosed with hereditary spherocytosis on convalescent blood films. On further questioning, a family history of hereditary spherocytosis was eventually revealed. The twins' maternal grandmother was known to have the condition asymptomatically. Their mother had prior to this never been tested, but later bloods would reveal a compatible biochemical picture.

  5. Acute parvovirus B19 infection in identical twins unmasking previously unidentified hereditary spherocytosis.

    PubMed

    Forde, Donall G; Cope, Alison; Stone, Ben

    2014-01-01

    Identical Caucasian male twins, previously fit, presented 1 week apart with short histories of fever and lethargy. The twins were febrile at presentation with profound pancytopaenia and evidence of haemolysis. There was no rash or arthralgia. Both required multiple red cell transfusions. The twins had positive IgM serology for Epstein-Barr virus (EBV), cytomegalovirus (CMV) and parvovirus B19. EBV viral capsid antigen and Epstein-Barr nuclear antigen IgGs were also positive however, suggesting past EBV exposure. Parvovirus B19 DNA was detected from peripheral blood PCR; CMV and EBV DNA PCRs were negative. Convalescent serology demonstrated no evolution of the CMV serological response, that is no IgG to CMV developed which implies an initial non-specific polyclonal IgM response. The twins recovered fully over 7 days, the first with a course of prednisolone and the second spontaneously. They were diagnosed with hereditary spherocytosis on convalescent blood films. On further questioning, a family history of hereditary spherocytosis was eventually revealed. The twins' maternal grandmother was known to have the condition asymptomatically. Their mother had prior to this never been tested, but later bloods would reveal a compatible biochemical picture. PMID:25073523

  6. Murine genotype influences the specificity, magnitude and persistence of murine mercury-induced autoimmunity.

    PubMed

    Hultman, P; Turley, S J; Eneström, S; Lindh, U; Pollard, K M

    1996-04-01

    Genetic factors are major contributors in determining the susceptibility to systemic autoimmune diseases. We studied the influence of genotype on systemic autoimmunity by treating female mice of the H-2s strains SJL/N, SJL/J, A.SW, and B10.S with mercuric chloride (HgCl2) for 10 weeks and then following autoantibody and tissue immune deposits during the subsequent 12 months. All strains developed antinucleolar antibodies (ANoA) of the IgG class which reacted in immunoblotting with a 34-kDa nucleolar protein identified as fibrillarin. The titre of ANoA attained after 10 weeks' treatment varied from 1:1,280 to 1:20,480 in the order: A.SW > SJL > > B10.S. Following cessation of HgCl2 treatment ANoA and antifibrillarin antibodies (AFA) persisted for up to 12 months, although some B10.S mice showed pronounced reduction not only of their autoantibody titres, but also systemic immune deposits when compared to other H-2s strains. A second set of autoantibodies targeted chromatin and in some mice specifically histones, and were distinguished from the ANoA by a rapid decline after treatment and a susceptibility linked to the non-H-2 genes of the SJL. Tissue levels of mercury remained elevated above untreated controls throughout the study period, suggesting that the mercury detected in lymphoid tissues may provide stimulation of lymphoid cells specific for fibrillarin for a considerable period after exposure has ceased. We conclude that H-2 as well as non-H-2 genetic factors distinctly influence not only the susceptibility to induction of autoimmunity, but also the specificity and magnitude of the response.

  7. Quantitation of human parvovirus B19 DNA in erythema infectiosum and aplastic crisis.

    PubMed

    Ishikawa, Aki; Yoto, Yuko; Tsugawa, Takeshi; Tsutsumi, Hiroyuki

    2014-12-01

    Several publications concerning the methods of real-time PCR for human parvovirus B19 (B19V) have appeared and some case reports mention B19V DNA loads. However, no large-scale study quantitating levels of B19V DNA in common or representative B19V manifestations such as erythema infectiosum and aplastic crisis has been performed. Consequently, using the TaqMan PCR assay, the B19V load in a large sample of subjects with erythema infectiosum or aplastic crisis was quantitated. Sixty-five subjects in the acute phase of erythema infectiosum were involved, and in addition 22 serum samples from seven subjects with B19V-associated aplastic crisis complicating chronic hemolytic anemia were also analyzed. In the acute phase of erythema infectiosum the median B19V DNA load in the serum samples from the acute phase of erythema infectiosum was 7.63 × 10(5)  genomes/ml, (range from 4.48 × 10(3) to 8.31 × 10(6)  genomes/ml). The serum B19V DNA load during the acute phase of aplastic crisis complicating chronic hemolytic anemia was extremely high, that is 10(10) -10(13)  genomes/ml, and decreased gradually to around 10(5) genomes/ml over 1-2 months. Although all subjects followed an almost uniform and typical clinical course of erythema infectiosum, there was a large individual variation of B19V DNA loads, that is differences of over 1,000 times. Extremely high B19V loads were observed in subjects with aplastic crisis. This study is the first large scale report of studies of the B19V DNA loads in subjects with erythema infectiosum and aplastic crisis, the most common and significant clinical manifestations by B19V infections.

  8. A test of genotypic variation in specificity of herbivore-induced responses in Solidago altissima L. (Asteraceae).

    PubMed

    Uesugi, Akane; Poelman, Erik H; Kessler, André

    2013-12-01

    Plant-induced responses to multiple herbivores can mediate ecological interactions among herbivore species, thereby influencing herbivore community composition in nature. Several studies have indicated high specificity of induced responses to different herbivore species. In addition, there may be genetic variation for plant response specificity that can have significant ecological implications, by altering the competitive strength and hierarchical relationships among interacting herbivore species. However, few studies have examined whether plant populations harbor genetic variation for induction specificity. Using three distinct genotypes of Solidago altissima plants, we examined whether specialist herbivore species Dichomeris leuconotella, Microrhopala vittata, and Trirhabda virgata elicit specific induction responses from plants (specificity of elicitation), and whether induction differentially affects these herbivore species (specificity of effect). Results from bioassays and secondary metabolite analyses suggest that there is specificity of both elicitation and effect in the induced responses: D. leuconotella and M. vittata preferred and performed better on leaves damaged by conspecifics than heterospecifics, and induced qualitatively different secondary metabolite profiles. In contrast, T. virgata equally avoided but physiologically tolerated all types of damage. These patterns of specificity suggest that plant-induced responses mediate asymmetric competitive interactions between herbivore species, which potentially intensifies inter-specific relative to intra-specific competition. Plant genotypes widely differed in overall susceptibility to the herbivores and secondary metabolite production, yet we found no genotype-by-treatment interactions in insect performance, preference and plant secondary metabolite production. This lack of genetic variation for induction specificity suggests that competitive interactions between herbivore species on S. altissima are

  9. Development of a Novel Allele-Specific PCR Method for Rapid Assessment of Nervous Necrosis Virus Genotypes.

    PubMed

    Toubanaki, Dimitra K; Margaroni, Maritsa; Karagouni, Evdokia

    2015-11-01

    Viral nervous necrosis infections are causing severe problems on aquaculture industry due to ecological and economic impacts. Their causal agent is nervous necrosis virus or nodavirus, which has been classified into four genotypes. Different genotypes correlate with differences in viral pathogenicity. Therefore, rational development of effective vaccines and diagnostic reagents requires analysis of the genetic variation. The development and validation of a polymerase chain reaction amplification (PCR)-based methodology for nodavirus genotype assessment in a simple and robust format is described. Degenerate external primers and two genotype-specific internal primers were utilized for simultaneous amplification of nodavirus products in a single PCR. A first set of cycles produced a long PCR product, defined by the outer primers, and the internal primers amplified short DNA fragments specific for each genotype in lower annealing temperature. Detection was based on the size of the short products. Nodavirus infected and healthy samples were analyzed and none of the non-infected samples showed any bands, while all infected samples were positive. The proposed method can be performed within 4 h and consumes standard PCR and electrophoresis reagents, with costs lower than 2€ per sample. Tetra-primer PCR is a suitable alternative for virus sequencing in medium scale research laboratories and farming facilities. PMID:26210900

  10. Development of RT-qPCR assays for the specific identification of two major genotypes of avian infectious bronchitis virus.

    PubMed

    Marandino, Ana; Tomás, Gonzalo; Hernández, Martín; Panzera, Yanina; Craig, María Isabel; Vagnozzi, Ariel; Vera, Federico; Techera, Claudia; Grecco, Sofía; Banda, Alejandro; Hernández, Diego; Pérez, Ruben

    2016-09-01

    Infectious bronchitis virus (Gammacoronavirus, Coronaviridae) is a genetically variable RNA virus (27.6kb) that causes one of the most persistent respiratory disease in poultry. The virus is classified in genotypes with different epidemiological relevance and clinical implications. The present study reports the development and validation of specific RT-qPCR assays for the detection of two major IBV genotypes: South America I (SAI) and Asia/South America II (A/SAII). The SAI genotype is an exclusive and widespread South American lineage while the A/SAII genotype is distributed in Asia, Europe and South America. Both identification assays employ TaqMan probes that hybridize with unique sequences in the spike glycoprotein gene. The assays successfully detected all the assessed strains belonging to both genotypes, showing high specificity and absence of cross-reactivity. Using serial dilutions of in vitro-transcribed RNA we obtained acceptable determination coefficients, PCR efficiencies and relatively small intra- and inter-assay variability. The assays demonstrated a wide dynamic range between 10(1)-10(7) and 10(2)-10(7) RNA copies/reaction for SAI and A/SAII strains, respectively. The possibility to characterize a large number of samples in a rapid, sensitive and reproducible way makes these techniques suitable tools for routine testing, IBV control, and epidemiological research in poultry.

  11. Development of a Novel Allele-Specific PCR Method for Rapid Assessment of Nervous Necrosis Virus Genotypes.

    PubMed

    Toubanaki, Dimitra K; Margaroni, Maritsa; Karagouni, Evdokia

    2015-11-01

    Viral nervous necrosis infections are causing severe problems on aquaculture industry due to ecological and economic impacts. Their causal agent is nervous necrosis virus or nodavirus, which has been classified into four genotypes. Different genotypes correlate with differences in viral pathogenicity. Therefore, rational development of effective vaccines and diagnostic reagents requires analysis of the genetic variation. The development and validation of a polymerase chain reaction amplification (PCR)-based methodology for nodavirus genotype assessment in a simple and robust format is described. Degenerate external primers and two genotype-specific internal primers were utilized for simultaneous amplification of nodavirus products in a single PCR. A first set of cycles produced a long PCR product, defined by the outer primers, and the internal primers amplified short DNA fragments specific for each genotype in lower annealing temperature. Detection was based on the size of the short products. Nodavirus infected and healthy samples were analyzed and none of the non-infected samples showed any bands, while all infected samples were positive. The proposed method can be performed within 4 h and consumes standard PCR and electrophoresis reagents, with costs lower than 2€ per sample. Tetra-primer PCR is a suitable alternative for virus sequencing in medium scale research laboratories and farming facilities.

  12. Evolutionary fate of rhizome-specific genes in a non-rhizomatous Sorghum genotype.

    PubMed

    Jang, C S; Kamps, T L; Tang, H; Bowers, J E; Lemke, C; Paterson, A H

    2009-03-01

    What is the fate of organ-specific genes after the organ is lost? For Sorghum propinquum and Sorghum halepense genes that were previously shown to have rhizome-enriched expression, we have conducted comparative analysis of both coding regions and regulatory sequences in Sorghum bicolor (non-rhizomatousness) and S. propinquum (rhizomatousness). Most genes with rhizome-enriched expression appear to have similar numbers of paralogous copies in both genotypes, with only three of 24 genes studied showing significant differences in copy numbers. We detected no greater propensity for mutation in S. bicolor than in S. propinquum of genes with rhizome-enriched expression in the latter. Several cis-acting regulatory elements, particularly an Myb-binding core (AACGG) that is involved in the regulation of the mitotic cyclin, were more abundant in promoters of S. propinquum than in non-rhizomatous S. bicolor or Oryza sativa (rice). We suggest that many genes with rhizome-enriched expression in S. propinquum may serve multiple functions, with partial loss of some of these functions in S. bicolor but ongoing purifying selection acting to preserve the remaining functions. Expressed genes in polyploid S. halepense rhizomes appeared to be more frequently derived from the S. propinquum than the S. bicolor progenitor, but there was some evidence of formation of novel alleles and 'recruitment' of S. bicolor genes to rhizome-enriched expression in S. halepense, suggesting that polyploidy may have offered new evolutionary potential to S. halepense.

  13. Investigating genotype specific response in photosynthetic behavior under drought stress and nitrogen limitation in Brassica rapa.

    NASA Astrophysics Data System (ADS)

    Pleban, J. R.; Mackay, D. S.; Ewers, B. E.; Weinig, C.; Aston, T.

    2015-12-01

    Challenges in terrestrial ecosystem modeling include characterizing the impact of stress on vegetation and the heterogeneous behavior of different species within the environment. In an effort to address these challenges the impacts of drought and nutrient limitation on the CO2 assimilation of multiple genotypes of Brassica rapa was investigated using the Farquhar Model (FM) of photosynthesis following a Bayesian parameterization and updating scheme. Leaf gas exchange and chlorophyll fluorescence measurements from an unstressed group (well-watered/well-fertilized) and two stressed groups (drought/well-fertilized and well-watered/nutrient limited) were used to estimate FM model parameters. Unstressed individuals were used to initialize Bayesian parameter estimation. Posterior mean estimates yielded a close fit with data as observed assimilation (An) closely matched predicted (Ap) with mean standard error for all individuals ranging from 0.8 to 3.1 μmol CO2 m-2 s-1. Posterior parameter distributions of the unstressed individuals were combined and fit to distributions to establish species level Bayesian priors of FM parameters for testing stress responses. Species level distributions of unstressed group identified mean maximum rates of carboxylation standardized to 25° (Vcmax25) as 101.8 μmol m-2 s-1 (± 29.0) and mean maximum rates of electron transport standardized to 25° (Jmax25) as 319.7 μmol m-2 s-1 (± 64.4). These updated priors were used to test the response of drought and nutrient limitations on assimilation. In the well-watered/nutrient limited group a decrease of 28.0 μmol m-2 s-1 was observed in mean estimate of Vcmax25, a decrease of 27.9 μmol m-2 s-1 in Jmax25 and a decrease in quantum yield from 0.40 mol photon/mol e- in unstressed individuals to 0.14 in the nutrient limited group. In the drought/well-fertilized group a decrease was also observed in Vcmax25 and Jmax25. The genotype specific unstressed and stressed responses were then used to

  14. Enterocytozoon bieneusi in sika deer (Cervus nippon) and red deer (Cervus elaphus): deer specificity and zoonotic potential of ITS genotypes.

    PubMed

    Zhao, Wei; Zhang, Weizhe; Wang, Rongjun; Liu, Weishi; Liu, Aiqin; Yang, Dong; Yang, Fengkun; Karim, Md Robiul; Zhang, Longxian

    2014-11-01

    As the most common cause of the human microsporidiosis, Enterocytozoon bieneusi has been found in a wide variety of animal hosts. Deers are the ruminant mammals living in a variety of biomes, and the distribution of deer species differ by geography. To understand the prevalence of natural infection of E. bieneusi in deer and to assess their epidemiological role in the transmission of microsporidiosis caused by E. bieneusi, 91 fecal specimens were collected from 86 sika deers and five red deers in the northeast of China. By PCR and sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene of E. bieneusi, an average infection rate of 31.9% (29/91) was observed in deer, with 32.6% (28/86) for sika deer, and 20% (1/5) for red deer. Six ITS genotypes were identified: one known genotype BEB6 (n = 20) and five novel genotypes HLJD-I to HLJD-IV (one each) and HLJD-V (n = 5). A phylogenetic analysis based on a neighbor-joining tree of the ITS gene sequences of E. bieneusi indicated that genotypes HLJD-II and HLJD-III fell into group 1 of zoonotic potential, while the other genotypes (BEB6, HLJD-I, HLJD-IV, HLJD-V) were clustered into so-called bovine-specific group 2. This is the first report of E. bieneusi in deer in China. The observation of genotype BEB6 in humans previously and in deer here and also the findings of the two novel genotypes (HLJD-II to HLJ-III) belonging to potential zoonotic group 1 suggested the possibility of deer in the transmission of E. bieneusi to humans.

  15. Enterocytozoon bieneusi in sika deer (Cervus nippon) and red deer (Cervus elaphus): deer specificity and zoonotic potential of ITS genotypes.

    PubMed

    Zhao, Wei; Zhang, Weizhe; Wang, Rongjun; Liu, Weishi; Liu, Aiqin; Yang, Dong; Yang, Fengkun; Karim, Md Robiul; Zhang, Longxian

    2014-11-01

    As the most common cause of the human microsporidiosis, Enterocytozoon bieneusi has been found in a wide variety of animal hosts. Deers are the ruminant mammals living in a variety of biomes, and the distribution of deer species differ by geography. To understand the prevalence of natural infection of E. bieneusi in deer and to assess their epidemiological role in the transmission of microsporidiosis caused by E. bieneusi, 91 fecal specimens were collected from 86 sika deers and five red deers in the northeast of China. By PCR and sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene of E. bieneusi, an average infection rate of 31.9% (29/91) was observed in deer, with 32.6% (28/86) for sika deer, and 20% (1/5) for red deer. Six ITS genotypes were identified: one known genotype BEB6 (n = 20) and five novel genotypes HLJD-I to HLJD-IV (one each) and HLJD-V (n = 5). A phylogenetic analysis based on a neighbor-joining tree of the ITS gene sequences of E. bieneusi indicated that genotypes HLJD-II and HLJD-III fell into group 1 of zoonotic potential, while the other genotypes (BEB6, HLJD-I, HLJD-IV, HLJD-V) were clustered into so-called bovine-specific group 2. This is the first report of E. bieneusi in deer in China. The observation of genotype BEB6 in humans previously and in deer here and also the findings of the two novel genotypes (HLJD-II to HLJ-III) belonging to potential zoonotic group 1 suggested the possibility of deer in the transmission of E. bieneusi to humans. PMID:25185666

  16. Characterization Based on the 56-Kda Type-Specific Antigen Gene of Orientia tsutsugamushi Genotypes Isolated from Leptotrombidium Mites and the Rodent Host Post-Infection

    PubMed Central

    Takhampunya, Ratree; Tippayachai, Bousaraporn; Promsathaporn, Sommai; Leepitakrat, Surachai; Monkanna, Taweesak; Schuster, Anthony L.; Melendrez, Melanie C.; Paris, Daniel H.; Richards, Allen L.; Richardson, Jason H.

    2014-01-01

    Characterization of the 56-kDa type-specific antigen (TSA) genes of Orientia tsutsugamushi (OT) from three naturally infected, laboratory-reared mite colonies comprising three species (Leptotrombidium deliense [Ld], Leptotrombidium imphalum [Li], and Leptotrombidium chiangraiensis [Lc]) has revealed the presence of single and coexisting OT genotypes found in individual chiggers. The Karp genotype was found in all of the chiggers examined, whereas Gilliam and UT302 genotypes were only observed in combination with the Karp genotype. From analysis of these OT genotypes after transmission from chiggers to mice it was determined that with the Lc and Li mites, the OT genotype composition in the rodent spleens post-infection had not changed and therefore resembled that observed in the feeding chiggers. However, only the Karp genotype was found in rodents after feeding by Ld chiggers carrying Karp and Gilliam genotypes. The current findings reveal a complex association among the host, pathogen, and vector. PMID:24297814

  17. Epidemiologic study of human parvovirus B19 infection in East China.

    PubMed

    Zhang, Lahong; Cai, Chengsong; Pan, Feng; Hong, Liquan; Luo, Xian; Hu, Sha; Xu, Jiali; Chen, Zhaojun

    2016-07-01

    Human parvovirus B19 (B19V) infection causes a number of diseases in humans, and, in some circumstances, can be life threatening. To understand the epidemiology of B19V infection in the greater metropolitan area of Hangzhou, East China, we performed surveys of IgM and IgG antibodies against B19V and quantification of B19V DNA, by using enzyme-linked immunosorbent assay and quantitative PCR, respectively, in plasma samples from diverse groups. These groups included anemia patients, Mycoplasma pneumonia- and Treponema pallidum-infected patients, HIV-positive individuals, and healthy blood donor volunteers. Our results demonstrated a low level of B19V IgG antibody presence, ranging from 21.9% to 41.8% in all the groups tested, suggesting a low prevalence of B19V infection in the area. Of note, we found that two healthy blood donors and one Mycoplasma pneumonia-infected patient had B19V IgM antibody among 1,290 plasma samples tested. The Mycoplasma pneumonia-infected patient had viremia with viral genome copies of 2.86 × 10(6) per ml of plasma. We detected a high rate of B19V DNA (7.1%) in HIV-positive injection drug users. Importantly, an amino acid mutation of P558S in the large non-structural protein NS1 was identified to be conserved among 14 B19V isolates from the HIV-positive group but not in the B19V isolate of the Mycoplasma pneumonia-infected patient, representing a hallmark of B19V isolates that circulate in HIV1-positive patients in the greater metropolitan area of Hangzhou, East China.

  18. Epidemiologic study of human parvovirus B19 infection in East China.

    PubMed

    Zhang, Lahong; Cai, Chengsong; Pan, Feng; Hong, Liquan; Luo, Xian; Hu, Sha; Xu, Jiali; Chen, Zhaojun

    2016-07-01

    Human parvovirus B19 (B19V) infection causes a number of diseases in humans, and, in some circumstances, can be life threatening. To understand the epidemiology of B19V infection in the greater metropolitan area of Hangzhou, East China, we performed surveys of IgM and IgG antibodies against B19V and quantification of B19V DNA, by using enzyme-linked immunosorbent assay and quantitative PCR, respectively, in plasma samples from diverse groups. These groups included anemia patients, Mycoplasma pneumonia- and Treponema pallidum-infected patients, HIV-positive individuals, and healthy blood donor volunteers. Our results demonstrated a low level of B19V IgG antibody presence, ranging from 21.9% to 41.8% in all the groups tested, suggesting a low prevalence of B19V infection in the area. Of note, we found that two healthy blood donors and one Mycoplasma pneumonia-infected patient had B19V IgM antibody among 1,290 plasma samples tested. The Mycoplasma pneumonia-infected patient had viremia with viral genome copies of 2.86 × 10(6) per ml of plasma. We detected a high rate of B19V DNA (7.1%) in HIV-positive injection drug users. Importantly, an amino acid mutation of P558S in the large non-structural protein NS1 was identified to be conserved among 14 B19V isolates from the HIV-positive group but not in the B19V isolate of the Mycoplasma pneumonia-infected patient, representing a hallmark of B19V isolates that circulate in HIV1-positive patients in the greater metropolitan area of Hangzhou, East China. PMID:26705119

  19. A Genotypic-Oriented View of CFTR Genetics Highlights Specific Mutational Patterns Underlying Clinical Macrocategories of Cystic Fibrosis

    PubMed Central

    Lucarelli, Marco; Bruno, Sabina Maria; Pierandrei, Silvia; Ferraguti, Giampiero; Stamato, Antonella; Narzi, Fabiana; Amato, Annalisa; Cimino, Giuseppe; Bertasi, Serenella; Quattrucci, Serena; Strom, Roberto

    2015-01-01

    Cystic fibrosis (CF) is a monogenic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The genotype–phenotype relationship in this disease is still unclear, and diagnostic, prognostic and therapeutic challenges persist. We enrolled 610 patients with different forms of CF and studied them from a clinical, biochemical, microbiological and genetic point of view. Overall, there were 125 different mutated alleles (11 with novel mutations and 10 with complex mutations) and 225 genotypes. A strong correlation between mutational patterns at the genotypic level and phenotypic macrocategories emerged. This specificity appears to largely depend on rare and individual mutations, as well as on the varying prevalence of common alleles in different clinical macrocategories. However, 19 genotypes appeared to underlie different clinical forms of the disease. The dissection of the pathway from the CFTR mutated genotype to the clinical phenotype allowed to identify at least two components of the variability usually found in the genotype–phenotype relationship. One component seems to depend on the genetic variation of CFTR, the other component on the cumulative effect of variations in other genes and cellular pathways independent from CFTR. The experimental dissection of the overall biological CFTR pathway appears to be a powerful approach for a better comprehension of the genotype–phenotype relationship. However, a change from an allele-oriented to a genotypic-oriented view of CFTR genetics is mandatory, as well as a better assessment of sources of variability within the CFTR pathway. PMID:25910067

  20. Detection of cytomegalovirus, human parvovirus B19, and herpes simplex virus-1/2 in women with first-trimester spontaneous abortions.

    PubMed

    Zhou, Ya; Bian, Guohui; Zhou, Qiongxiu; Gao, Zhan; Liao, Pu; Liu, Yu; He, Miao

    2015-10-01

    The relationship between viral infections and first-trimester spontaneous abortions is not well-understood. The study aim was to investigate the prevalence of cytomegalovirus (CMV), human parvovirus B19 (B19V), and herpes simplex virus-1/2 (HSV-1/2) infection by molecular and serological techniques in women experiencing spontaneous miscarriage in the first trimester of pregnancy. Plasma samples were examined for CMV, B19V, and HSV-1/2 DNA using real-time quantitative polymerase chain reaction (Real-time qPCR), and for specific IgG antibodies against B19V, CMV, and HSV-1/2 using serological assays. The abortion group consisted of women (n = 1,716) with a history of two or more first-trimester spontaneous abortions. Women younger than 30 years possess higher portion to experience spontaneous abortion. No specimens were positive for B19V or CMV DNA. Seven out of the 1,716 specimens were positive for HSV-1/2 DNA. By serology, 47.24% of patients were positive for B19V IgG, 39.66% for HSV IgG, 79.31% for CMV IgG, and 9.31% for B19V IgM. The high rate of positivity for CMV IgG suggests that the majority of women with first-trimester spontaneous abortions are not susceptible to primary CMV infection. The lack of virus DNA in the majority of cases indicates that B19V, CMV, and HSV-1/2 infection is not commonly associated with first-trimester spontaneous abortion.

  1. Enhanced specificity of TPMT*2 genotyping using unidirectional wild-type and mutant allele-specific scorpion primers in a single tube.

    PubMed

    Chen, Dong; Yang, Zhao; Xia, Han; Huang, Jun-Fu; Zhang, Yang; Jiang, Tian-Nun; Wang, Gui-Yu; Chuai, Zheng-Ran; Fu, Wei-Ling; Huang, Qing

    2014-01-01

    Genotyping of thiopurine S-methyltransferase (TPMT) is recommended for predicting the adverse drug response of thiopurines. In the current study, a novel version of allele-specific PCR (AS-PCR), termed competitive real-time fluorescent AS-PCR (CRAS-PCR) was developed to analyze the TPMT*2 genotype in ethnic Chinese. This technique simultaneously uses wild-type and mutant allele-specific scorpion primers in a single reaction. To determine the optimal conditions for both traditional AS-PCR and CRAS-PCR, we used the Taguchi method, an engineering optimization process that balances the concentrations of all components using an orthogonal array rather than a factorial array. Instead of running up to 264 experiments with the conventional factorial method, the Taguchi method achieved the same optimization using only 16 experiments. The optimized CRAS-PCR system completely avoided non-specific amplification occurring in traditional AS-PCR and could be performed at much more relaxed reaction conditions at 1% sensitivity, similar to traditional AS-PCR. TPMT*2 genotyping of 240 clinical samples was consistent with published data. In conclusion, CRAS-PCR is a novel and robust genotyping method, and the Taguchi method is an effective tool for the optimization of molecular analysis techniques. PMID:24705376

  2. Enhanced Specificity of TPMT*2 Genotyping Using Unidirectional Wild-Type and Mutant Allele-Specific Scorpion Primers in a Single Tube

    PubMed Central

    Chen, Dong; Yang, Zhao; Xia, Han; Huang, Jun-Fu; Zhang, Yang; Jiang, Tian-Nun; Wang, Gui-Yu; Chuai, Zheng-Ran; Fu, Wei-Ling; Huang, Qing

    2014-01-01

    Genotyping of thiopurine S-methyltransferase (TPMT) is recommended for predicting the adverse drug response of thiopurines. In the current study, a novel version of allele-specific PCR (AS-PCR), termed competitive real-time fluorescent AS-PCR (CRAS-PCR) was developed to analyze the TPMT*2 genotype in ethnic Chinese. This technique simultaneously uses wild-type and mutant allele-specific scorpion primers in a single reaction. To determine the optimal conditions for both traditional AS-PCR and CRAS-PCR, we used the Taguchi method, an engineering optimization process that balances the concentrations of all components using an orthogonal array rather than a factorial array. Instead of running up to 264 experiments with the conventional factorial method, the Taguchi method achieved the same optimization using only 16 experiments. The optimized CRAS-PCR system completely avoided non-specific amplification occurring in traditional AS-PCR and could be performed at much more relaxed reaction conditions at 1% sensitivity, similar to traditional AS-PCR. TPMT*2 genotyping of 240 clinical samples was consistent with published data. In conclusion, CRAS-PCR is a novel and robust genotyping method, and the Taguchi method is an effective tool for the optimization of molecular analysis techniques. PMID:24705376

  3. Genotype-specific mutations in the polymerase gene of hepatitis B virus potentially associated with resistance to oral antiviral therapy.

    PubMed

    Mirandola, Silvia; Sebastiani, Giada; Rossi, Cristina; Velo, Emanuela; Erne, Elke Maria; Vario, Alessandro; Tempesta, Diego; Romualdi, Chiara; Campagnolo, Davide; Alberti, Alfredo

    2012-12-01

    The evolution of hepatitis B virus (HBV) and the role of different variants during antiviral therapy may be influenced by HBV genotype. We have therefore analysed substitutions potentially related to nucleos(t)ide analogues (NAs) resistance at 42 positions within RT-region in a cohort of patients with chronic hepatitis B in relation to HBV-genotype. RT mutations analysis was performed by direct sequencing in 200 NAs-naïve patients and in 64 LAM or LAM+ADV experienced patients with NAs resistance, infected mainly by HBV-genotypes D and A. 27 polymorphic-sites were identified among the 42 positions analysed and 64 novel mutations were detected in 23 positions. Genotype-D displayed the highest mutation frequency (6.4%) among all HBV-genotypes analysed. Single or multiple mutations were detected in 80% of naïve patients. Overall, the most frequent single mutations were at residues rt54, rt53 and rt91 which may associate with significantly lower HBV-DNA levels (p=0.001). Comparison with sequencing data of patients failing LMV or LAM+ADV therapy revealed an higher frequency of novel genotype-specific mutations if compared with naïve patients: 3 mutations under LAM monotherapy in HBV-D (rtS85F; rtL91I; rtC256G) and 3 mutations under ADV therapy in HBV-A (rtI53V; rtW153R; rtF221Y). In HBV-D treated patients the dominant resistance mutation was rtL80V (31.4%) and rtM204I (60%) in LAM+ADV group while LAM-treated patients showed a preference of rtM204V (51.9%). Interestingly, none of HBV-A patients had mutation rtM204I under ADV add-on treatment but all of them had the "V" AA substitution. These results suggested that in patients with CHB, HBV-genotype might be relevant in the evolution and development of drug resistance showing also different mutation patterns in the YMDD motif between HBV genotype D and A. PMID:23026293

  4. Genotype-specific variation in West Nile virus dispersal in California.

    PubMed

    Duggal, Nisha K; Reisen, William K; Fang, Ying; Newman, Ruchi M; Yang, Xiao; Ebel, Gregory D; Brault, Aaron C

    2015-11-01

    West Nile virus (WNV) is an arbovirus that was first reported in North America in New York in 1999 and, by 2003, had spread more than 4000 km to California. However, variation in viral genetics associated with spread is not well understood. Herein, we report sequences for more than 100 WNV isolates made from mosquito pools that were collected from 2003 to 2011 as part of routine surveillance by the California Mosquito-borne Virus Surveillance System. We performed phylogeographic analyses and demonstrated that 5 independent introductions of WNV (1 WN02 genotype strain and 4 SW03 genotype strains) occurred in California. The SW03 genotype of WNV was constrained to the southwestern U.S. and had a more rapid rate of spread. In addition, geographic constraint of WNV strains within a single region for up to 6 years suggest viral maintenance has been driven by resident, rather than migratory, birds and overwintering in mosquitoes.

  5. Parvovirus B19 infection in five European countries: seroepidemiology, force of infection and maternal risk of infection

    PubMed Central

    MOSSONG, J.; HENS, N.; FRIEDERICHS, V.; DAVIDKIN, I.; BROMAN, M.; LITWINSKA, B.; SIENNICKA, J.; TRZCINSKA, A.; VAN DAMME, P.; BEUTELS, P.; VYSE, A.; SHKEDY, Z.; AERTS, M.; MASSARI, M.; GABUTTI, G.

    2008-01-01

    SUMMARY We conducted a seroprevalence survey in Belgium, Finland, England & Wales, Italy and Poland on 13 449 serum samples broadly representative in terms of geography and age. Samples were tested for the presence of immunoglobulin G antibody using an enzyme immunoassay. The age-specific risk of infection was estimated using parametric and non-parametric statistical modelling. The age-specific risk in all five countries was highest in children aged 7–9 years and lower in adults. The average proportion of women of child-bearing age susceptible to parvovirus B19 infection and the risk of a pregnant women acquiring B19 infection during pregnancy was estimated to be 26% and 0·61% in Belgium, 38% and 0·69% in England & Wales, 43·5% and 1·24% in Finland, 39·9% and 0·92% in Italy and 36·8% and 1·58% in Poland, respectively. Our study indicates substantial epidemiological differences in Europe regarding parvovirus B19 infection. PMID:17956642

  6. Combining quantitative trait loci analysis with physiological models to predict genotype-specific transpiration rates.

    PubMed

    Reuning, Gretchen A; Bauerle, William L; Mullen, Jack L; McKay, John K

    2015-04-01

    Transpiration is controlled by evaporative demand and stomatal conductance (gs ), and there can be substantial genetic variation in gs . A key parameter in empirical models of transpiration is minimum stomatal conductance (g0 ), a trait that can be measured and has a large effect on gs and transpiration. In Arabidopsis thaliana, g0 exhibits both environmental and genetic variation, and quantitative trait loci (QTL) have been mapped. We used this information to create a genetically parameterized empirical model to predict transpiration of genotypes. For the parental lines, this worked well. However, in a recombinant inbred population, the predictions proved less accurate. When based only upon their genotype at a single g0 QTL, genotypes were less distinct than our model predicted. Follow-up experiments indicated that both genotype by environment interaction and a polygenic inheritance complicate the application of genetic effects into physiological models. The use of ecophysiological or 'crop' models for predicting transpiration of novel genetic lines will benefit from incorporating further knowledge of the genetic control and degree of independence of core traits/parameters underlying gs variation.

  7. RNA-Seq reveals genotype-specific molecular responses to water deficit in eucalyptus

    PubMed Central

    2011-01-01

    Background In a context of climate change, phenotypic plasticity provides long-lived species, such as trees, with the means to adapt to environmental variations occurring within a single generation. In eucalyptus plantations, water availability is a key factor limiting productivity. However, the molecular mechanisms underlying the adaptation of eucalyptus to water shortage remain unclear. In this study, we compared the molecular responses of two commercial eucalyptus hybrids during the dry season. Both hybrids differ in productivity when grown under water deficit. Results Pyrosequencing of RNA extracted from shoot apices provided extensive transcriptome coverage - a catalog of 129,993 unigenes (49,748 contigs and 80,245 singletons) was generated from 398 million base pairs, or 1.14 million reads. The pyrosequencing data enriched considerably existing Eucalyptus EST collections, adding 36,985 unigenes not previously represented. Digital analysis of read abundance in 14,460 contigs identified 1,280 that were differentially expressed between the two genotypes, 155 contigs showing differential expression between treatments (irrigated vs. non irrigated conditions during the dry season), and 274 contigs with significant genotype-by-treatment interaction. The more productive genotype displayed a larger set of genes responding to water stress. Moreover, stress signal transduction seemed to involve different pathways in the two genotypes, suggesting that water shortage induces distinct cellular stress cascades. Similarly, the response of functional proteins also varied widely between genotypes: the most productive genotype decreased expression of genes related to photosystem, transport and secondary metabolism, whereas genes related to primary metabolism and cell organisation were over-expressed. Conclusions For the most productive genotype, the ability to express a broader set of genes in response to water availability appears to be a key characteristic in the maintenance

  8. Effects of human parvovirus B19 VP1 unique region protein on macrophage responses

    PubMed Central

    Tzang, Bor-Show; Chiu, Chun-Ching; Tsai, Chun-Chou; Lee, Yi-Ju; Lu, I-Jung; Shi, Jing-Yu; Hsu, Tsai-Ching

    2009-01-01

    Background Activity of secreted phospholipase A (sPLA2) has been implicated in a wide range of cellular responses. However, little is known about the function of human parvovirus B19-VP1 unique region (VP1u) with sPLA2 activity on macrophage. Methods To investigate the roles of B19-VP1u in response to macrophage, phospholipase A2 activity, cell migration assay, phagocytosis activity, metalloproteinase assay, RT-PCR and immunoblotting were performed. Results In the present study, we report that migration, phagocytosis, IL-6, IL-1β mRNA, and MMP9 activity are significantly increased in RAW264.7 cells by B19-VP1u protein with sPLA2 activity, but not by B19-VP1uD175A protein that is mutated and lacks sPLA2 activity. Additionally, significant increases of phosphorylated ERK1/2 and JNK proteins were detected in macrophages that were treated with B19-VP1u protein, but not when they were treated with B19-VP1uD175A protein. Conclusion Taken together, our experimental results suggest that B19-VP1u with sPLA2 activity affects production of IL-6, IL-1β mRNA, and MMP9 activity, possibly through the involvement of ERK1/2 and JNK signaling pathways. These findings could provide clues in understanding the role of B19-VP1u and its sPLA2 enzymatic activity in B19 infection and B19-related diseases. PMID:19272185

  9. Quantitation of parvovirus B19 DNA sequences by competitive PCR: differential hybridization of the amplicons and immunoenzymatic detection on microplate.

    PubMed

    Gallinella, G; Zerbini, M; Musiani, M; Venturoli, S; Gentilomi, G; Manaresi, E

    1997-04-01

    A competitive PCR assay was developed to quantify B19 DNA sequences. Target and internal standard sequences were co-amplified by the same set of primers. The internal standard competitor was constructed by recombinant PCR and differed from the original genome sequence in a 21-bp mutagenized fragment, internal to the region amplified by the same set of primers. The internal standard competitor was cloned in a plasmid vector and the cloned fragment used in all the experiments. Target and internal standard sequences were labelled with digoxigenin during the co-amplification reaction and the different amplicons were detected in two separate hybridization reactions by biotinylated probes specific for the original 21-bp sequence or the mutagenized one. Hybridized amplicons were captured onto streptavidin-oated microtitre wells and detected by anti-digoxigenin antibodies conjugated to peroxidase. The chromogenic reaction for peroxidase was quantitatively evaluated by optical density determination. The titration curve subsequently developed showed a linear relationship over the range 10(2) to 10(5) genome copies, thus obtaining an exact quantitative evaluation over a wide range together with good sensitivity. Nine reference serum samples positive for B19 DNA and eight negative serum samples were tested by the competitive PCR assay for the quantitation of B19 DNA sequences.

  10. Identification of the major structural and nonstructural proteins encoded by human parvovirus B19 and mapping of their genes by procaryotic expression of isolated genomic fragments.

    PubMed

    Cotmore, S F; McKie, V C; Anderson, L J; Astell, C R; Tattersall, P

    1986-11-01

    Plasma from a child with homozygous sickle-cell disease, sampled during the early phase of an aplastic crisis, contained human parvovirus B19 virions. Plasma taken 10 days later (during the convalescent phase) contained both immunoglobulin M and immunoglobulin G antibodies directed against two viral polypeptides with apparent molecular weights of 83,000 and 58,000 which were present exclusively in the particulate fraction of the plasma taken during the acute phase. These two protein species comigrated at 110S on neutral sucrose velocity gradients with the B19 viral DNA and thus appear to constitute the viral capsid polypeptides. The B19 genome was molecularly cloned into a bacterial plasmid vector. Restriction endonuclease fragments of this cloned B19 genome were treated with BAL 31 and shotgun cloned into the open reading frame expression vector pJS413. Two expression constructs containing B19 sequences from different halves of the viral genome were obtained, which directed the synthesis, in bacteria, of segments of virally encoded protein. These polypeptide fragments were then purified and used to immunize rabbits. Antibodies against a protein sequence specified between nucleotides 2897 and 3749 recognized both the 83- and 58-kilodalton capsid polypeptides in aplastic plasma taken during the acute phase and detected similar proteins in the tissues of a stillborn fetus which had been infected transplacentally with B19. Antibodies against a protein sequence encoded in the other half of the B19 genome (nucleotides 1072 through 2044) did not react specifically with any protein in plasma taken during the acute phase but recognized three nonstructural polypeptides of 71, 63, and 52 kilodaltons present in the liver and, at lower levels, in some other tissues of the transplacentally infected fetus.

  11. Genotype-Specific Interaction of Latent TGFβ Binding Protein 4 with TGFβ

    PubMed Central

    Lamar, Kay-Marie; Miller, Tamari; Dellefave-Castillo, Lisa; McNally, Elizabeth M.

    2016-01-01

    Latent TGFβ binding proteins are extracellular matrix proteins that bind latent TGFβ to form the large latent complex. Nonsynonymous polymorphisms in LTBP4, a member of the latent TGFβ binding protein gene family, have been linked to several human diseases, underscoring the importance of TGFβ regulation for a range of phenotypes. Because of strong linkage disequilibrium across the LTBP4 gene, humans have two main LTBP4 alleles that differ at four amino acid positions, referred to as IAAM and VTTT for the encoded residues. VTTT is considered the “risk” allele and associates with increased intracellular TGFβ signaling and more deleterious phenotypes in muscular dystrophy and other diseases. We now evaluated LTBP4 nsSNPs in dilated cardiomyopathy, a distinct disorder associated with TGFβ signaling. We stratified based on self-identified ethnicity and found that the LTBP4 VTTT allele is associated with increased risk of dilated cardiomyopathy in European Americans extending the diseases that associate with LTBP4 genotype. However, the association of LTBP4 SNPs with dilated cardiomyopathy was not observed in African Americans. To elucidate the mechanism by which LTBP4 genotype exerts this differential effect, TGFβ’s association with LTBP4 protein was examined. LTBP4 protein with the IAAM residues bound more latent TGFβ compared to the LTBP4 VTTT protein. Together these data provide support that LTBP4 genotype exerts its effect through differential avidity for TGFβ accounting for the differences in TGFβ signaling attributed to these two alleles. PMID:26918958

  12. Genotype-Specific Interaction of Latent TGFβ Binding Protein 4 with TGFβ.

    PubMed

    Lamar, Kay-Marie; Miller, Tamari; Dellefave-Castillo, Lisa; McNally, Elizabeth M

    2016-01-01

    Latent TGFβ binding proteins are extracellular matrix proteins that bind latent TGFβ to form the large latent complex. Nonsynonymous polymorphisms in LTBP4, a member of the latent TGFβ binding protein gene family, have been linked to several human diseases, underscoring the importance of TGFβ regulation for a range of phenotypes. Because of strong linkage disequilibrium across the LTBP4 gene, humans have two main LTBP4 alleles that differ at four amino acid positions, referred to as IAAM and VTTT for the encoded residues. VTTT is considered the "risk" allele and associates with increased intracellular TGFβ signaling and more deleterious phenotypes in muscular dystrophy and other diseases. We now evaluated LTBP4 nsSNPs in dilated cardiomyopathy, a distinct disorder associated with TGFβ signaling. We stratified based on self-identified ethnicity and found that the LTBP4 VTTT allele is associated with increased risk of dilated cardiomyopathy in European Americans extending the diseases that associate with LTBP4 genotype. However, the association of LTBP4 SNPs with dilated cardiomyopathy was not observed in African Americans. To elucidate the mechanism by which LTBP4 genotype exerts this differential effect, TGFβ's association with LTBP4 protein was examined. LTBP4 protein with the IAAM residues bound more latent TGFβ compared to the LTBP4 VTTT protein. Together these data provide support that LTBP4 genotype exerts its effect through differential avidity for TGFβ accounting for the differences in TGFβ signaling attributed to these two alleles.

  13. The pan-genotype specificity of the hepatitis C virus anti-core monoclonal antibody C7-50 is contingent on the quasispecies profile of a population.

    PubMed

    Palmer, Brendan A; Menton, John; Levis, John; Kenny-Walsh, Elizabeth; Crosbie, Orla; Fanning, Liam J

    2012-11-01

    The inter/intra-genotype quasispecies makeup of hepatitis C virus (HCV) has retarded the development of antibodies capable of pan-genotype reactivity. Mutations, even in conserved domains, are tolerated to a degree. In this report, we characterise the pan-genotype specificity of the commercially available monoclonal anti-HCV core antibody C7-50. We demonstrate the antibody's ability to detect HCV core protein following infection of Huh7 cells with serum-derived HCV of genotypes 2-5 and that a single-site polymorphism in a genotype 3a core amino acid sequence is sufficient to disrupt antibody recognition of the epitope. This same polymorphism is a feature of genotype 3 viruses. PMID:22828781

  14. Childhood Adversity Increases Risk for Nicotine Dependence and Interacts with α5 Nicotinic Acetylcholine Receptor Genotype Specifically in Males

    PubMed Central

    Xie, Pingxing; Kranzler, Henry R; Zhang, Huiping; Oslin, David; Anton, Raymond F; Farrer, Lindsay A; Gelernter, Joel

    2012-01-01

    The relative importance of specific genetic and environmental factors in regulating nicotine dependence (ND) risk, including the effects on specific forms of childhood adversity on smoking risk, have been understudied. Genome-wide association studies and rodent models have demonstrated that the α5 nicotinic acetylcholine receptor gene (CHRNA5) is important in regulating nicotine intake. Childhood adversity increases the methylation level of the CHRNA5 promoter region in European Americans (EAs), an effect that was observed only in males (Zhang et al, submitted for publication). In view of this potential sex difference in the effects of early life experience on smoking, we investigated the presence of a sex-specific gene-by-environment effect of this marker on ND risk. A nonsynonymous SNP in CHRNA5 previously associated to ND and several related traits, rs16969968, was genotyped in 2206 EAs (1301 men and 905 women). The main and interactive effects of childhood adversity and rs16969968 genotype on diagnosis of ND and ND defined by dichotomized Fagerstrom test for ND (FTND) scores were explored. Men and women were analyzed separately to test for sex differences. Childhood adversity significantly increased ND risk in both sexes, and the effect in women was twice than that in men. Significant interactive effects of childhood adversity and rs16969968 genotype were observed in men (ND: OR=1.80, 95% CI=1.18–2.73, P=0.0044; FTND: OR=1.79, 95% CI=1.11–2.88, P=0.012). No interaction was found in women. This study provides evidence of a sex-specific gene × environment effect of CHRNA5 and childhood adversity on the risk for ND. PMID:22012472

  15. Risk of perinatal transmission of rubella and parvovirus B19 in Jordanian pregnant women.

    PubMed

    Bdour, Salwa

    2006-04-12

    The seroprevalence and the risk of perinatal transmission of rubella virus (RV) and human parvovirus B19 were assessed in 439 Jordanian pregnant women. Seroprevalence data indicate that 43.7%, 48.7% and 19% of women are susceptible to RV, parvovirus B19 infections and both, respectively. A total of 12.2% and 20.2% of RV susceptible women are at the first and second trimester of gestation, respectively. They are at risk of bearing infants with congenital rubella syndrome (CRS). However, 15% and 23% of parvovirus B19 susceptible women are at the first and second trimester of gestation, respectively. They are at risk of fetal loss or hydrops fetalis. Prenatal screening for these viruses, postpartum MMR vaccination and parvovirus B19 passive immunization are recommended. PMID:16460842

  16. The "pressure pan" evolution of human erythrovirus B19 in the Amazon, Brazil.

    PubMed

    Barros de Freitas, Ronaldo; Durigon, Edison Luiz; Oliveira, Darleise de Souza; Romano, Camila Malta; Castro de Freitas, Maria Rute; Linhares, Alexandre da Costa; Melo, Fernando Lucas; Walshkeller, Lílian; Barbosa, Maria Luisa; Huatuco, Egma M Mayta; Holmes, Edward C; Zanotto, Paolo Marinho de A

    2007-12-20

    To understand the evolutionary dynamics of human parvovirus B19, we analyzed VP1 and VP2 gene sequences of B19 sampled from Belém (Amazon), the city of São Paulo, Brazil and globally. Our analysis revealed a strikingly different pattern of evolutionary change for those viral lineages introduced into Belém, which exhibited a higher rate of nonsynonymous substitutions compared to those viruses sampled from other locations. We propose that difference this is due to the high prevalence of B19 in Belém (up to 85%) compared to other locations (prevalences of approximately 50%), which imposes a more intense selection pressure. Hence, those B19 lineages introduced into Belém experienced an elevated rate of amino acid change, driven by positive selection, in order to generate serial re-infections in a small web of transmission, which can be thought of as an evolutionary "pressure pan".

  17. Interaction of parvovirus B19 with human erythrocytes alters virus structure and cell membrane integrity.

    PubMed

    Bönsch, Claudia; Kempf, Christoph; Ros, Carlos

    2008-12-01

    The unique region of the capsid protein VP1 (VP1u) of B19 virus (B19V) elicits a dominant immune response and has a phospholipase A(2) (PLA(2)) activity required for the infection. Despite these properties, we have observed that the VP1u-PLA(2) motif occupies an internal position in the capsid. However, brief exposure to increasing temperatures induced a progressive accessibility of the PLA(2) motif as well as a proportional increase of the PLA(2) activity. Similarly, upon binding on human red blood cells (RBCs), a proportion of the capsids externalized the VP1u-PLA(2) motif. Incubation of B19V with RBCs from 17 healthy donors resulted in extensive virus attachment ranging between 3,000 and 30,000 virions per cell. B19V empty capsids represent an important fraction of the viral particles circulating in the blood (30 to 40%) and bind to RBCs in the same way as full capsids. The extensive B19V binding to RBCs did not cause direct hemolysis but an increased osmotic fragility of the cells by a mechanism involving the PLA(2) activity of the exposed VP1u. Analysis of a blood sample from an individual with a recent B19V infection revealed that, at this particular moment of the infection, the virions circulating in the blood were mostly associated to the RBC fraction. However, the RBC-bound B19V was not able to infect susceptible cells. These observations indicate that RBCs play a significant role during B19V infection by triggering the exposure of the immunodominant VP1u including its PLA(2) constituent. On the other hand, the early exposure of VP1u might facilitate viral internalization and/or uncoating in target cells.

  18. Interaction of Parvovirus B19 with Human Erythrocytes Alters Virus Structure and Cell Membrane Integrity▿

    PubMed Central

    Bönsch, Claudia; Kempf, Christoph; Ros, Carlos

    2008-01-01

    The unique region of the capsid protein VP1 (VP1u) of B19 virus (B19V) elicits a dominant immune response and has a phospholipase A2 (PLA2) activity required for the infection. Despite these properties, we have observed that the VP1u-PLA2 motif occupies an internal position in the capsid. However, brief exposure to increasing temperatures induced a progressive accessibility of the PLA2 motif as well as a proportional increase of the PLA2 activity. Similarly, upon binding on human red blood cells (RBCs), a proportion of the capsids externalized the VP1u-PLA2 motif. Incubation of B19V with RBCs from 17 healthy donors resulted in extensive virus attachment ranging between 3,000 and 30,000 virions per cell. B19V empty capsids represent an important fraction of the viral particles circulating in the blood (30 to 40%) and bind to RBCs in the same way as full capsids. The extensive B19V binding to RBCs did not cause direct hemolysis but an increased osmotic fragility of the cells by a mechanism involving the PLA2 activity of the exposed VP1u. Analysis of a blood sample from an individual with a recent B19V infection revealed that, at this particular moment of the infection, the virions circulating in the blood were mostly associated to the RBC fraction. However, the RBC-bound B19V was not able to infect susceptible cells. These observations indicate that RBCs play a significant role during B19V infection by triggering the exposure of the immunodominant VP1u including its PLA2 constituent. On the other hand, the early exposure of VP1u might facilitate viral internalization and/or uncoating in target cells. PMID:18815302

  19. Model structure analysis to estimate basic immunological processes and maternal risk for parvovirus B19

    PubMed Central

    Goeyvaerts, Nele; Hens, Niel; Aerts, Marc; Beutels, Philippe

    2011-01-01

    After a steep monotone rise with age, the seroprevalence profiles for human parvovirus B19 (PVB19) display a decrease or plateau between the ages of 20 and 40, in each of 5 European countries. We investigate whether this phenomenon is induced by waning antibodies for PVB19 and, if this is the case, whether secondary infections are plausible, or whether boosting may occur. Several immunological scenarios are tested for PVB19 by fitting different compartmental dynamic transmission models to serological data using data on social contact patterns. The social contact approach has already been shown informative to estimate transmission rates and the basic reproduction number for infections transmitted predominantly through nonsexual social contacts. Our results show that for 4 countries, model selection criteria favor the scenarios allowing for waning immunity at an age-specific rate over the assumption of lifelong immunity, assuming that the transmission rates are directly proportional to the contact rates. Different views on the evolution of the immune response to PVB19 infection lead to altered estimates of the age-specific force of infection and the basic reproduction number. The scenarios which allow for multiple infections during one lifetime predict a higher frequency of PVB19 infection in pregnant women and of associated fetal deaths. When prevaccination serological data are available, the framework developed in this paper could prove worthwhile to investigate these different scenarios for other infections as well, such as cytomegalovirus. PMID:20841333

  20. Impact of genotype-specific herd immunity on the circulatory dynamism of norovirus: a 10-year longitudinal study of viral acute gastroenteritis.

    PubMed

    Sakon, Naomi; Yamazaki, Kenji; Nakata, Keiko; Kanbayashi, Daiki; Yoda, Tomoko; Mantani, Masanobu; Kase, Tetsuo; Takahashi, Kazuo; Komano, Jun

    2015-03-15

    Human norovirus is a major cause of viral acute gastroenteritis worldwide. However, the transition of endemic norovirus genotypes remains poorly understood. The characteristics of natural immunity against norovirus are unclear because few studies have been performed in the natural infection setting. This prospective 10-year surveillance study of acute gastroenteritis in the province of Osaka, Japan, revealed that norovirus spread shows temporal, geographic, and age group-specific features in the humans. Genogroup II genotype 4 (GII.4) was detected in most sporadic pediatric cases, as well as in foodborne and nursing home outbreaks, respectively. The dominant genotypes in outbreaks at childcare facilities and schools shifted every season and involved GI, GII.2, GII.3, GII.4, and GII.6. Evidence at both the facility and individual levels indicated that genotype-specific herd immunity lasted long enough to influence the endemic norovirus genotype in the next season. Thus, norovirus circulates through human populations in a uniquely dynamic fashion.

  1. The VP1-unique region of parvovirus B19 induces myocardial injury in mice.

    PubMed

    Nie, Xiaojing; Zhang, Guocheng; Xu, Dongliang; Sun, Xin; Li, Zhihong; Li, Xiaoqing; Zhang, Xuehong; He, Fei; Li, Yunming

    2010-01-01

    As a common human pathogen, parvovirus B19 (B19V) has been shown to be associated with many heart diseases, such as myocarditis, cardiomyopathy and cardiopericarditis. The virus protein 1-unique region (VP1u) is critical to B19V infectivity, but its role in the pathogenesis of B19V-induced myocardial injury has not been well studied. In this study to investigate the effects ofVP1u on the host myocardium, we first expressed a recombinant VP1u protein in Escherichia coli, produced it on a large scale by high-volume fermentation, and purified it using the AKTA explorer 100 system. Following treatment of mice with the recombinant protein, we then examined changes in the morphology of the cardiac muscles, the titre of anti-VP1u protein antibodies, and a panel of heart functional protein markers. Our results show that VP1u alone is sufficient to elicit pathological and ultrastructural changes in the host myocardium, and to increase the levels of the functional enzymes aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine kinase (CK), creatine kinase isoenzyme (CK-MB) and alpha-hydroxybutyric acid dehydrogenase (alpha-HBDH). The changes in myocardial pathology and myocardial zymogram indicate that the VP1u protein of B19V causes myocardial injury, and may largely contribute to the pathogenesis of B19V-induced heart diseases.

  2. Identification of self-incompatibility genotypes of apricot (Prunus armeniaca L.) by S-allele-specific PCR analysis.

    PubMed

    Jie, Qi; Shupeng, Gai; Jixiang, Zhang; Manru, Gu; Huairui, Shu

    2005-08-01

    A cDNA of 417 bp encoding an S-RNase gene, named PA S3, was isolated from apricot, Prunus aremeniaca. Nine S-alleles, S1-S9, were recognized by S-allele-specific PCR and confirmed by Southern blot analysis using PA S3 as probe. The S-genotypes of the six cultivars were determined and the results of self- and cross-pollination tests among the six cultivars were consistent with the predicted S-haplotypes by PCR analysis.

  3. Type-specific PCR assays for Babesia bovis msa-1 genotypes in Asia: Revisiting the genetic diversity in Sri Lanka, Mongolia, and Vietnam.

    PubMed

    Liyanagunawardena, Nilukshi; Sivakumar, Thillaiampalam; Kothalawala, Hemal; Silva, Seekkuge Susil Priyantha; Battsetseg, Badgar; Lan, Dinh Thi Bich; Inoue, Noboru; Igarashi, Ikuo; Yokoyama, Naoaki

    2016-01-01

    Babesia bovis is the most virulent Babesia organism, resulting in a high mortality rate in cattle. The genetic diversity of B. bovis merozoite surface antigens (MSAs), such as MSA-1, MSA-2b, and MSA-2c, might be linked to altered immune profiles in the host animals. The present study aimed to develop type-specific PCR assays for Asian msa-1 genotypes, thereby re-analyzing the genetic diversity of msa-1 in Sri Lanka, Mongolia, and Vietnam. Specific primers were designed for nine Asian msa-1 genotypes, which had been detected based on the phylogeny constructed using msa-1 gene sequences retrieved from the GenBank database. Specificity of the type-specific PCR assays was confirmed using plasmids containing the inserts of msa-1 gene fragments that represent Asian genotypes. Furthermore, no amplicons were observed by these PCR assays when DNA samples of Babesia bigemina, Babesia ovata, Theileria annulata, Theileria orientalis, Trypanosoma evansi, Trypanosoma theileri, Anaplasma marginale, and Anaplasma bovis, and non-infected bovine blood were analyzed. In total, 109 B. bovis-positive blood DNA samples sourced from Sri Lanka (44 cattle), Mongolia (26 cattle), and Vietnam (23 cattle and 16 water buffaloes) were then screened by the type-specific PCR assays. The sequences derived from all of the PCR amplicons were phylogenetically analyzed. Out of 109 DNA samples, 23 (20 from cattle and 3 from water buffaloes) were positive for at least one genotype. In agreement with previous studies, five and four different genotypes were detected among the DNA samples from Sri Lanka and Vietnam, respectively. In contrast, four genotypes, including three novel genotypes, were detected from Mongolia. Five DNA samples were found to be co-infected with multiple genotypes. The sequences of the PCR amplicons clustered phylogenetically within the corresponding clades. These findings indicated that the type-specific PCR assays described herein are useful for the determination of genotypic

  4. Type-specific PCR assays for Babesia bovis msa-1 genotypes in Asia: Revisiting the genetic diversity in Sri Lanka, Mongolia, and Vietnam.

    PubMed

    Liyanagunawardena, Nilukshi; Sivakumar, Thillaiampalam; Kothalawala, Hemal; Silva, Seekkuge Susil Priyantha; Battsetseg, Badgar; Lan, Dinh Thi Bich; Inoue, Noboru; Igarashi, Ikuo; Yokoyama, Naoaki

    2016-01-01

    Babesia bovis is the most virulent Babesia organism, resulting in a high mortality rate in cattle. The genetic diversity of B. bovis merozoite surface antigens (MSAs), such as MSA-1, MSA-2b, and MSA-2c, might be linked to altered immune profiles in the host animals. The present study aimed to develop type-specific PCR assays for Asian msa-1 genotypes, thereby re-analyzing the genetic diversity of msa-1 in Sri Lanka, Mongolia, and Vietnam. Specific primers were designed for nine Asian msa-1 genotypes, which had been detected based on the phylogeny constructed using msa-1 gene sequences retrieved from the GenBank database. Specificity of the type-specific PCR assays was confirmed using plasmids containing the inserts of msa-1 gene fragments that represent Asian genotypes. Furthermore, no amplicons were observed by these PCR assays when DNA samples of Babesia bigemina, Babesia ovata, Theileria annulata, Theileria orientalis, Trypanosoma evansi, Trypanosoma theileri, Anaplasma marginale, and Anaplasma bovis, and non-infected bovine blood were analyzed. In total, 109 B. bovis-positive blood DNA samples sourced from Sri Lanka (44 cattle), Mongolia (26 cattle), and Vietnam (23 cattle and 16 water buffaloes) were then screened by the type-specific PCR assays. The sequences derived from all of the PCR amplicons were phylogenetically analyzed. Out of 109 DNA samples, 23 (20 from cattle and 3 from water buffaloes) were positive for at least one genotype. In agreement with previous studies, five and four different genotypes were detected among the DNA samples from Sri Lanka and Vietnam, respectively. In contrast, four genotypes, including three novel genotypes, were detected from Mongolia. Five DNA samples were found to be co-infected with multiple genotypes. The sequences of the PCR amplicons clustered phylogenetically within the corresponding clades. These findings indicated that the type-specific PCR assays described herein are useful for the determination of genotypic

  5. Genotype-specific changes in vitamin B6 content and the PDX family in potato.

    PubMed

    Mooney, Sutton; Chen, Liyuan; Kühn, Christina; Navarre, Roy; Knowles, N Richard; Hellmann, Hanjo

    2013-01-01

    Vitamin B6 is one of the most versatile cofactors in plants and an essential phytonutrient in the human diet that benefits a variety of human health aspects. Although biosynthesis of the vitamin has been well resolved in recent years, the main research is currently based on Arabidopsis thaliana with very little work done on major crop plants. Here we provide the first report on interactions and expression profiles of PDX genes for vitamin B6 biosynthesis in potato and how vitamin B6 content varies in tubers of different genotypes. The results demonstrate that potato is an excellent resource for this vitamin and that strong natural variation in vitamin B6 content among the tested cultivars indicates high potential to fortify vitamin B6 nutrition in potato-based foods.

  6. Mapping IS6110 in high-copy number Mycobacterium tuberculosis strains shows specific insertion points in the Beijing genotype

    PubMed Central

    2013-01-01

    Background Mycobacterium tuberculosis Beijing strains are characterized by a large number of IS6110 copies, suggesting the potential implication of this element in the virulence and capacity for rapid dissemination characteristic of this family. This work studies the insetion points of IS6110 in high-copy clinical isolates specifically focusing on the Beijing genotype. Results In the present work we mapped the insertion points of IS6110 in all the Beijing strains available in the literature and in the DNA sequence databases. We generated a representative primer collection of the IS6110 locations, which was used to analyse 61 high-copy clinical isolates. A total of 440 points of insertion were identified and analysis of their flanking regions determined the exact location, the direct repeats (DRs), the orientation and the distance to neighboring genes of each copy of IS6110. We identified specific points of insertion in Beijing strains that enabled us to obtain a dendrogram that groups the Beijing genotype. Conclusions This work presents a detailed analysis of locations of IS6110 in high-copy clinical isolates, showing points of insertion present with high frequency in the Beijing family and absent in other strains. PMID:23800083

  7. Identification of the major structural and nonstructural proteins encoded by human parvovirus B19 and mapping of their genes by procaryotic expression of isolated genomic fragments

    SciTech Connect

    Cotmore, S.F.; McKie, V.C.; Anderson, L.J.; Astell, C.R.; Tattersall, P.

    1986-11-01

    Plasma from a child with homozygous sickle-cell disease, sampled during the early phase of an aplastic crisis, contained human parvovirus B19 virions. Plasma taken 10 days later (during the convalescent phase) contained both immunoglobulin M and immunoglobulin G antibodies directed against two viral polypeptides with apparent molecular weights for 83,000 and 58,000 which were present exclusively in the particulate fraction of the plasma taken during the acute phase. These two protein species comigrated at 110S on neutral sucrose velocity gradients with the B19 viral DNA and thus appear to constitute the viral capsid polypeptides. The B19 genome was molecularly cloned into a bacterial plasmid vector. Two expression constructs containing B19 sequences from different halves of the viral genome were obtained, which directed the synthesis, in bacteria, of segments of virally encoded protein. These polypeptide fragments were then purified and used to immunize rabbits. Antibodies against a protein sequence specified between nucleotides 2897 and 3749 recognized both the 83- and 58-kilodalton capsid polypeptides in aplastic plasma taken during the acute phase and detected similar proteins in the similar proteins in the tissues of a stillborn fetus which had been infected transplacentally with B19. Antibodies against a protein sequence encoded in the other half of the B19 genome (nucleotides 1072 through 2044) did not react specifically with any protein in plasma taken during the acute phase but recognized three nonstructural polypeptides of 71, 63, and 52 kilodaltons present in the liver and, at lower levels, in some other tissues of the transplacentally infected fetus.

  8. High-throughput identification of genotype-specific cancer vulnerabilities in mixtures of barcoded tumor cell lines.

    PubMed

    Yu, Channing; Mannan, Aristotle M; Yvone, Griselda Metta; Ross, Kenneth N; Zhang, Yan-Ling; Marton, Melissa A; Taylor, Bradley R; Crenshaw, Andrew; Gould, Joshua Z; Tamayo, Pablo; Weir, Barbara A; Tsherniak, Aviad; Wong, Bang; Garraway, Levi A; Shamji, Alykhan F; Palmer, Michelle A; Foley, Michael A; Winckler, Wendy; Schreiber, Stuart L; Kung, Andrew L; Golub, Todd R

    2016-04-01

    Hundreds of genetically characterized cell lines are available for the discovery of genotype-specific cancer vulnerabilities. However, screening large numbers of compounds against large numbers of cell lines is currently impractical, and such experiments are often difficult to control. Here we report a method called PRISM that allows pooled screening of mixtures of cancer cell lines by labeling each cell line with 24-nucleotide barcodes. PRISM revealed the expected patterns of cell killing seen in conventional (unpooled) assays. In a screen of 102 cell lines across 8,400 compounds, PRISM led to the identification of BRD-7880 as a potent and highly specific inhibitor of aurora kinases B and C. Cell line pools also efficiently formed tumors as xenografts, and PRISM recapitulated the expected pattern of erlotinib sensitivity in vivo. PMID:26928769

  9. Parvovirus B19 Passive Transmission by Transfusion of Intercept® Blood System-Treated Platelet Concentrate

    PubMed Central

    Gowland, Peter; Fontana, Stefano; Stolz, Martin; Andina, Nicola; Niederhauser, Christoph

    2016-01-01

    Summary Background Pathogen reduction methods for blood components are effective for a large number of viruses though less against small, non-enveloped viruses such as Parvovirus B19 (B19V). This article describes the passive transmission by transfusion of two B19V-contaminated pooled platelet concentrates (PCs) which were treated with the Intercept® blood pathogen reduction system. Case Reports Two transfusion cases of B19V-contaminated Intercept-treated pooled PCs were described. Due to the analysis delay, the PCs were already transfused. The viral content of each donation was 4.87 × 1010 IU/ml in case 1and 1.46 × 108 IU/ml in case 2. B19V (52 IU/ml) was detected in the recipient of the case 1 PC, whereas no virus could be detected in the case 2 PC recipient. A B19V IgM response and a transient boost of the underlying B19V IgG immune status and was observed in recipient 1. Recipient of the case 2 PC remained B19V IgG- and IgM-negative. B19V DNA sequence and phylogenetic analysis revealed a 100% homology between donor and recipient. Conclusion This report describes passive B19V transmission by a PC with very high B19 viral load which elicited a transient boost of the B19V immunity, but not by a PC with a lower B19V content, suggesting that there is a B19 viral load threshold value at which B19V inactivation is exceeded. PMID:27403092

  10. Transcriptomes of Eight Arabidopsis thaliana Accessions Reveal Core Conserved, Genotype- and Organ-Specific Responses to Flooding Stress1[OPEN

    PubMed Central

    van Veen, Hans; Vashisht, Divya; Akman, Melis; Girke, Thomas; Mustroph, Angelika; Reinen, Emilie; Kooiker, Maarten; van Tienderen, Peter; Voesenek, Laurentius A.C.J.

    2016-01-01

    Climate change has increased the frequency and severity of flooding events, with significant negative impact on agricultural productivity. These events often submerge plant aerial organs and roots, limiting growth and survival due to a severe reduction in light reactions and gas exchange necessary for photosynthesis and respiration, respectively. To distinguish molecular responses to the compound stress imposed by submergence, we investigated transcriptomic adjustments to darkness in air and under submerged conditions using eight Arabidopsis (Arabidopsis thaliana) accessions differing significantly in sensitivity to submergence. Evaluation of root and rosette transcriptomes revealed an early transcriptional and posttranscriptional response signature that was conserved primarily across genotypes, although flooding susceptibility-associated and genotype-specific responses also were uncovered. Posttranscriptional regulation encompassed darkness- and submergence-induced alternative splicing of transcripts from pathways involved in the alternative mobilization of energy reserves. The organ-specific transcriptome adjustments reflected the distinct physiological status of roots and shoots. Root-specific transcriptome changes included marked up-regulation of chloroplast-encoded photosynthesis and redox-related genes, whereas those of the rosette were related to the regulation of development and growth processes. We identified a novel set of tolerance genes, recognized mainly by quantitative differences. These included a transcriptome signature of more pronounced gluconeogenesis in tolerant accessions, a response that included stress-induced alternative splicing. This study provides organ-specific molecular resolution of genetic variation in submergence responses involving interactions between darkness and low-oxygen constraints of flooding stress and demonstrates that early transcriptome plasticity, including alternative splicing, is associated with the ability to cope

  11. Impaired genome encapsidation restricts the in vitro propagation of human parvovirus B19.

    PubMed

    Wolfisberg, Raphael; Ruprecht, Nico; Kempf, Christoph; Ros, Carlos

    2013-10-01

    The lack of a permissive cell culture system hampers the study of human parvovirus B19 (B19V). UT7/Epo is one of the few established cell lines that can be infected with B19V but generates none or few infectious progeny. Recently, hypoxic conditions or the use of primary CD36+ erythroid progenitor cells (CD36+ EPCs) have been shown to improve the infection. These novel approaches were evaluated in infection and transfection experiments. Hypoxic conditions or the use of CD36+ EPCs resulted in a significant acceleration of the infection/transfection and a modest increase in the yield of capsid progeny. However, under all tested conditions, genome encapsidation was impaired seriously. Further analysis of the cell culture virus progeny revealed that differently to the wild-type virus, the VP1 unique region (VP1u) was exposed partially and was unable to become further externalized upon heat treatment. The fivefold axes pore, which is used for VP1u externalization and genome encapsidation, might be constricted by the atypical VP1u conformation explaining the packaging failure. Although CD36+ EPCs and hypoxia facilitate B19V infection, large quantities of infectious progeny cannot be generated due to a failure in genome encapsidation, which arises as a major limiting factor for the in vitro propagation of B19V.

  12. Standardization of a PCR-ELISA in serum samples: diagnosis of active parvovirus B19 infection.

    PubMed

    Zerbini, M; Gallinella, G; Manaresi, E; Musiani, M; Gentilomi, G; Venturoli, S

    1999-10-01

    To standardize a PCR assay for the detection of parvovirus B19 DNA in serum samples three different sample treatments were evaluated on the basis of the efficiency of recovery, reproducibility, convenience of sample handling, and presence of PCR inhibitors. Moreover, the presence of an internal standard competitor as the working reagent at one defined concentration in a competitive PCR-ELISA has been suggested as a valid tool to standardize and validate the assay. The results indicated that serum sample treatment by rapid heating fulfilled the criteria for a routine practice in the diagnostic laboratory. Titration experiments carried out to define the optimal amount of the internal standard competitor to use in PCR-ELISA showed that at 2 x10(2) competitor copies, any amplification interferences between target and competitor sequences were avoided. The internal standard competitor in a competitive PCR-ELISA allows the detection of false-negative results due to PCR inhibitors in the samples or large amounts of target DNA. Heating treatment and competitive PCR-ELISA for the detection of parvovirus B19 DNA were applied to the testing of 347 serum samples, which were submitted to the laboratory for B19 investigation. Of the 34 serum samples that were positive for B19 DNA, 15 were from adult patients and 19 from pediatric subjects. B19 infection was associated with haematological disorders, nonimmunological foetal hydrops, atypical rash, arthropathies, hepatic dysfunction, nonspecific symptoms, and congenital infections.

  13. Genotype-Specific Variation in the Structure of Root Fungal Communities Is Related to Chickpea Plant Productivity

    PubMed Central

    Hamel, Chantal; Gan, Yantai; Tar'an, Bunyamin; Knight, Joan Diane

    2015-01-01

    Increasing evidence supports the existence of variations in the association of plant roots with symbiotic fungi that can improve plant growth and inhibit pathogens. However, it is unclear whether intraspecific variations in the symbiosis exist among plant cultivars and if they can be used to improve crop productivity. In this study, we determined genotype-specific variations in the association of chickpea roots with soil fungal communities and evaluated the effect of root mycota on crop productivity. A 2-year field experiment was conducted in southwestern Saskatchewan, the central zone of the chickpea growing region of the Canadian prairie. The effects of 13 cultivars of chickpea, comprising a wide range of phenotypes and genotypes, were tested on the structure of root-associated fungal communities based on internal transcribed spacer (ITS) and 18S rRNA gene markers using 454 amplicon pyrosequencing. Chickpea cultivar significantly influenced the structure of the root fungal community. The magnitude of the effect varied with the genotypes evaluated, and effects were consistent across years. For example, the roots of CDC Corrine, CDC Cory, and CDC Anna hosted the highest fungal diversity and CDC Alma and CDC Xena the lowest. Fusarium sp. was dominant in chickpea roots but was less abundant in CDC Corrine than the other cultivars. A bioassay showed that certain of these fungal taxa, including Fusarium species, can reduce the productivity of chickpea, whereas Trichoderma harzianum can increase chickpea productivity. The large variation in the profile of chickpea root mycota, which included growth-promoting and -inhibiting species, supports the possibility of improving the productivity of chickpea by improving its root mycota in chickpea genetic improvement programs using traditional breeding techniques. PMID:25616789

  14. Genotype-specific variation in the structure of root fungal communities is related to chickpea plant productivity.

    PubMed

    Bazghaleh, Navid; Hamel, Chantal; Gan, Yantai; Tar'an, Bunyamin; Knight, Joan Diane

    2015-04-01

    Increasing evidence supports the existence of variations in the association of plant roots with symbiotic fungi that can improve plant growth and inhibit pathogens. However, it is unclear whether intraspecific variations in the symbiosis exist among plant cultivars and if they can be used to improve crop productivity. In this study, we determined genotype-specific variations in the association of chickpea roots with soil fungal communities and evaluated the effect of root mycota on crop productivity. A 2-year field experiment was conducted in southwestern Saskatchewan, the central zone of the chickpea growing region of the Canadian prairie. The effects of 13 cultivars of chickpea, comprising a wide range of phenotypes and genotypes, were tested on the structure of root-associated fungal communities based on internal transcribed spacer (ITS) and 18S rRNA gene markers using 454 amplicon pyrosequencing. Chickpea cultivar significantly influenced the structure of the root fungal community. The magnitude of the effect varied with the genotypes evaluated, and effects were consistent across years. For example, the roots of CDC Corrine, CDC Cory, and CDC Anna hosted the highest fungal diversity and CDC Alma and CDC Xena the lowest. Fusarium sp. was dominant in chickpea roots but was less abundant in CDC Corrine than the other cultivars. A bioassay showed that certain of these fungal taxa, including Fusarium species, can reduce the productivity of chickpea, whereas Trichoderma harzianum can increase chickpea productivity. The large variation in the profile of chickpea root mycota, which included growth-promoting and -inhibiting species, supports the possibility of improving the productivity of chickpea by improving its root mycota in chickpea genetic improvement programs using traditional breeding techniques.

  15. Protocol: a simple gel-free method for SNP genotyping using allele-specific primers in rice and other plant species

    PubMed Central

    2010-01-01

    Background Genotype analysis using multiple single nucleotide polymorphisms (SNPs) is a useful but labor-intensive or high-cost procedure in plant research. Here we describe an alternative genotyping method that is suited to multi-sample or multi-locus SNP genotyping and does not require electrophoresis or specialized equipment. Results We have developed a simple method for multi-sample or multi-locus SNP genotyping using allele-specific primers (ASP). More specifically, we (1) improved the design of allele-specific primers, (2) established a method to detect PCR products optically without electrophoresis, and (3) standardized PCR conditions for parallel genomic assay using various allele-specific primers. As an illustration of multi-sample SNP genotyping using ASP, we mapped the locus for lodging resistance in a typhoon (lrt5). Additionally, we successfully tested multi-locus ASP-PCR analysis using 96 SNPs located throughout the genomes of rice (Oryza sativa) cultivars 'Koshihikari' and 'Kasalath', and demonstrated its applicability to other diverse cultivars/subspecies, including wild rice (O. rufipogon). Conclusion Our ASP methodology allows characterization of SNPs genotypes without electrophoresis, expensive probes or specialized equipment, and is highly versatile due to the flexibility in the design of primers. The method could be established easily in any molecular biology laboratory, and is applicable to diverse organisms. PMID:20409329

  16. Parasite-mediated protection against osmotic stress for Paramecium caudatum infected by Holospora undulata is host genotype specific.

    PubMed

    Duncan, Alison B; Fellous, Simon; Accot, Robin; Alart, Marie; Chantung Sobandi, Kevin; Cosiaux, Ariane; Kaltz, Oliver

    2010-11-01

    Under certain conditions, otherwise parasitic organisms may become beneficial to their host. Parasite-mediated heat and osmotic stress resistance have been demonstrated for Paramecium caudatum, infected by several species of parasitic bacteria of the genus Holospora. Here, using the micronucleus-specific bacterium Holospora undulata, we investigate how infection mediates the response of two genotypes (clones 'K8' and 'VEN') of P. caudatum to heat (35 °C) and osmotic (0.24% NaCl) stress. In contrast to previous findings, we find no evidence for heat stress protection in infected individuals. We do, however, show an effect of symbiont-mediated osmotic stress resistance for the K8 clone, with infected individuals having higher survival than their uninfected counterparts up to 24 h after the onset of salt exposure. Despite this, both infected and uninfected individuals of the VEN clone showed higher survival rates than clone K8 individuals under osmotic stress. Thus, it would seem that parasite-mediated stress protection is restricted to certain combinations of host genotypes and types of stress and does not represent a general phenomenon in this system.

  17. Complete remission of human parvovirus b19 associated symptoms by loxoprofen in patients with atopic predispositions.

    PubMed

    Kazama, Itsuro; Sasagawa, Naoko; Nakajima, Toshiyuki

    2012-01-01

    Two cases of women in their thirties with past histories of atopic dermatitis and allergic rhinitis developed a low grade fever, followed by a butterfly-shaped erythema, swelling of their fingers, and polyarthralgia. Despite such symptoms that overlap with those of systemic lupus erythematosus (SLE), the diagnostic criteria for SLE were not fulfilled. Due to positive results for human parvovirus B19 (HPV-B19) IgM antibodies in the serum, diagnoses of HPV-B19 infection were made in both cases. Although acetaminophen failed to improve their deteriorating symptoms, a nonsteroidal anti-inflammatory drug (NSAID), loxoprofen, completely removed the symptoms immediately after the administration. In those cases, since the patients were predisposed to atopic disorders, an increased immunological response based on the lymphocyte hypersensitivity was likely to be involved in the pathogenesis. The immunomodulatory property of NSAID was thought to repress such lymphocyte activity and thus provided a rapid and sustained remission of the disease. PMID:22611409

  18. The globoside receptor triggers structural changes in the B19 virus capsid that facilitate virus internalization.

    PubMed

    Bönsch, Claudia; Zuercher, Christoph; Lieby, Patricia; Kempf, Christoph; Ros, Carlos

    2010-11-01

    Globoside (Gb4Cer), Ku80 autoantigen, and α5β1 integrin have been identified as cell receptors/coreceptors for human parvovirus B19 (B19V), but their role and mechanism of interaction with the virus are largely unknown. In UT7/Epo cells, expression of Gb4Cer and CD49e (integrin alpha-5) was high, but expression of Ku80 was insignificant. B19V colocalized with Gb4Cer and, to a lesser extent, with CD49e. However, only anti-Gb4Cer antibodies could disturb virus attachment. Only a small proportion of cell-bound viruses were internalized, while the majority became detached from the receptor. When added to uninfected cells, the receptor-detached virus showed superior cell binding capacity and infectivity. Attachment of B19V to cells triggered conformational changes in the capsid leading to the accessibility of the N terminus of VP1 (VP1u) to antibodies, which was maintained in the receptor-detached virus. VP1u became similarly accessible to antibodies following incubation of B19V particles with increasing concentrations of purified Gb4Cer. The receptor-mediated exposure of VP1u is critical for virus internalization, since capsids lacking VP1 could bind to cells but were not internalized. Moreover, an antibody against the N terminus of VP1u disturbed virus internalization, but only when present during and not after virus attachment, indicating the involvement of this region in binding events required for internalization. These results suggest that Gb4Cer is not only the primary receptor for B19V attachment but also the mediator of capsid rearrangements required for subsequent interactions leading to virus internalization. The capacity of the virus to detach and reattach again would enhance the probability of productive infections.

  19. Characterization of the Early Steps of Human Parvovirus B19 Infection

    PubMed Central

    Quattrocchi, Silva; Ruprecht, Nico; Bönsch, Claudia; Bieli, Sven; Zürcher, Christoph; Boller, Klaus; Kempf, Christoph

    2012-01-01

    The early steps of human parvovirus B19 (B19V) infection were investigated in UT7/Epo cells. B19V and its receptor globoside (Gb4Cer) associate with lipid rafts, predominantly of the noncaveolar type. Pharmacological disruption of the lipid rafts inhibited infection when the drug was added prior to virus attachment but not after virus uptake. B19V is internalized by clathrin-dependent endocytosis and spreads rapidly throughout the endocytic pathway, reaching the lysosomal compartment within minutes, where a substantial proportion is degraded. B19V did not permeabilize the endocytic vesicles, indicating a mechanism of endosomal escape without apparent membrane damage. Bafilomycin A1 (BafA1) and NH4Cl, which raise endosomal pH, blocked the infection by preventing endosomal escape, resulting in a massive accumulation of capsids in the lysosomes. In contrast, in the presence of chloroquine (CQ), the transfer of incoming viruses from late endosomes to lysosomes was prevented; the viral DNA was not degraded; and the infection was boosted. In contrast to the findings for untreated or BafA1-treated cells, the viral DNA was progressively associated with the nucleus in CQ-treated cells, reaching a plateau by 3 h postinternalization, a time coinciding with the initiation of viral transcription. At this time, more than half of the total intracellular viral DNA was associated with the nucleus; however, the capsids remained extranuclear. Our studies provide the first insight into the early steps of B19V infection and reveal mechanisms involved in virus uptake, endocytic trafficking, and nuclear penetration. PMID:22718826

  20. The Globoside Receptor Triggers Structural Changes in the B19 Virus Capsid That Facilitate Virus Internalization▿

    PubMed Central

    Bönsch, Claudia; Zuercher, Christoph; Lieby, Patricia; Kempf, Christoph; Ros, Carlos

    2010-01-01

    Globoside (Gb4Cer), Ku80 autoantigen, and α5β1 integrin have been identified as cell receptors/coreceptors for human parvovirus B19 (B19V), but their role and mechanism of interaction with the virus are largely unknown. In UT7/Epo cells, expression of Gb4Cer and CD49e (integrin alpha-5) was high, but expression of Ku80 was insignificant. B19V colocalized with Gb4Cer and, to a lesser extent, with CD49e. However, only anti-Gb4Cer antibodies could disturb virus attachment. Only a small proportion of cell-bound viruses were internalized, while the majority became detached from the receptor. When added to uninfected cells, the receptor-detached virus showed superior cell binding capacity and infectivity. Attachment of B19V to cells triggered conformational changes in the capsid leading to the accessibility of the N terminus of VP1 (VP1u) to antibodies, which was maintained in the receptor-detached virus. VP1u became similarly accessible to antibodies following incubation of B19V particles with increasing concentrations of purified Gb4Cer. The receptor-mediated exposure of VP1u is critical for virus internalization, since capsids lacking VP1 could bind to cells but were not internalized. Moreover, an antibody against the N terminus of VP1u disturbed virus internalization, but only when present during and not after virus attachment, indicating the involvement of this region in binding events required for internalization. These results suggest that Gb4Cer is not only the primary receptor for B19V attachment but also the mediator of capsid rearrangements required for subsequent interactions leading to virus internalization. The capacity of the virus to detach and reattach again would enhance the probability of productive infections. PMID:20826697

  1. Measles, mumps, rubella, and human parvovirus B19 infections and neurologic disease.

    PubMed

    Bale, James F

    2014-01-01

    While the systemic disorders associated with measles, mumps, and rubella viruses and human parvovirus B19 tend to be mild, each virus can produce potentially life-threatening neurologic disease in human hosts, especially when these viruses infect young children. Two of the viruses, rubella and parvovirus B19, can be vertically transmitted to fetuses during maternal infection and cause congenital infection. Neurologic complications are common after intrauterine infection with the rubella virus, a condition known as the congenital rubella syndrome. Two, measles and rubella viruses, can induce "slow viral" infections, serious, disorders that can occur several years after the initial exposure to the virus and typically have fatal outcomes.

  2. [Virological diagnosis of an outbreak of fever and rashes caused by parvovirus B19, Cuba, 1995].

    PubMed

    Guzmán, M G; Rosario, D; Rodríguez, M E; Alvarez, M; Rodríguez, R; Oropesa, S; Laferté, J; Resik, S

    1997-01-01

    The results obtained in the study of an a outbreak of fever and rash occurred in Havana City in March, 1995, are reported. Dengue, measles, rubella, herpes simplex, and Epstein Barr were discarded as causal agents of the outbreak in the samples of 35 patients. Parvovirus B19 was identified as the causing agent of the outbreak by the detection of IgM antibodies and the polymerase chain reaction technique (PCR). The infection produced by this agent was confirmed in 14/18 samples (77.7%) by some of the techniques used. This study makes reference to the first outbreak of Parvovirus B19 that was proved in Cuba.

  3. Genotyping by Sequencing Using Specific Allelic Capture to Build a High-Density Genetic Map of Durum Wheat

    PubMed Central

    Holtz, Yan; Ardisson, Morgane; Ranwez, Vincent; Besnard, Alban; Leroy, Philippe; Poux, Gérard; Roumet, Pierre; Viader, Véronique; Santoni, Sylvain; David, Jacques

    2016-01-01

    Targeted sequence capture is a promising technology which helps reduce costs for sequencing and genotyping numerous genomic regions in large sets of individuals. Bait sequences are designed to capture specific alleles previously discovered in parents or reference populations. We studied a set of 135 RILs originating from a cross between an emmer cultivar (Dic2) and a recent durum elite cultivar (Silur). Six thousand sequence baits were designed to target Dic2 vs. Silur polymorphisms discovered in a previous RNAseq study. These baits were exposed to genomic DNA of the RIL population. Eighty percent of the targeted SNPs were recovered, 65% of which were of high quality and coverage. The final high density genetic map consisted of more than 3,000 markers, whose genetic and physical mapping were consistent with those obtained with large arrays. PMID:27171472

  4. Indications for both host-specific and introduced genotypes of Staphylococcus aureus in marine mammals.

    PubMed

    van Elk, Cornelis E; Boelens, Hélène A M; van Belkum, Alex; Foster, Geoffrey; Kuiken, Thijs

    2012-05-01

    Staphylococcus aureus is present in the marine environment and causes disease in marine mammals. To determine whether marine mammals are colonized by host-specific strains or by strains originating from other species, we performed multi-locus sequence typing on ten S. aureus strains isolated from marine mammals in the U.K., the Netherlands, and the Antarctic. Four new sequence types of S. aureus were discovered. S. aureus strains from a southern elephant seal (n=1) and harbour porpoises (n=2) did not cluster with known S. aureus strains, suggesting that they may be host species-specific. In contrast, S. aureus strains from harbour seals (n=3), other harbour porpoises (n=3), and a grey seal (n=1) clustered with S. aureus strains previously isolated from domestic ruminants, humans, or birds, suggesting that these S. aureus strains in marine mammals were introduced from terrestrial species. PMID:22112853

  5. SNPsplit: Allele-specific splitting of alignments between genomes with known SNP genotypes

    PubMed Central

    Krueger, Felix; Andrews, Simon R.

    2016-01-01

    Sequencing reads overlapping polymorphic sites in diploid mammalian genomes may be assigned to one allele or the other. This holds the potential to detect gene expression, chromatin modifications, DNA methylation or nuclear interactions in an allele-specific fashion. SNPsplit is an allele-specific alignment sorter designed to read files in SAM/BAM format and determine the allelic origin of reads or read-pairs that cover known single nucleotide polymorphic (SNP) positions. For this to work libraries must have been aligned to a genome in which all known SNP positions were masked with the ambiguity base 'N' and aligned using a suitable mapping program such as Bowtie2, TopHat, STAR, HISAT2, HiCUP or Bismark. SNPsplit also provides an automated solution to generate N-masked reference genomes for hybrid mouse strains based on the variant call information provided by the Mouse Genomes Project. The unique ability of SNPsplit to work with various different kinds of sequencing data including RNA-Seq, ChIP-Seq, Bisulfite-Seq or Hi-C opens new avenues for the integrative exploration of allele-specific data. PMID:27429743

  6. SNPsplit: Allele-specific splitting of alignments between genomes with known SNP genotypes.

    PubMed

    Krueger, Felix; Andrews, Simon R

    2016-01-01

    Sequencing reads overlapping polymorphic sites in diploid mammalian genomes may be assigned to one allele or the other. This holds the potential to detect gene expression, chromatin modifications, DNA methylation or nuclear interactions in an allele-specific fashion. SNPsplit is an allele-specific alignment sorter designed to read files in SAM/BAM format and determine the allelic origin of reads or read-pairs that cover known single nucleotide polymorphic (SNP) positions. For this to work libraries must have been aligned to a genome in which all known SNP positions were masked with the ambiguity base 'N' and aligned using a suitable mapping program such as Bowtie2, TopHat, STAR, HISAT2, HiCUP or Bismark. SNPsplit also provides an automated solution to generate N-masked reference genomes for hybrid mouse strains based on the variant call information provided by the Mouse Genomes Project. The unique ability of SNPsplit to work with various different kinds of sequencing data including RNA-Seq, ChIP-Seq, Bisulfite-Seq or Hi-C opens new avenues for the integrative exploration of allele-specific data. PMID:27429743

  7. Possible involvement of miRNAs in tropism of Parvovirus B19.

    PubMed

    Anbarlou, Azadeh; AkhavanRahnama, Mahshid; Atashi, Amir; Soleimani, Masoud; Arefian, Ehsan; Gallinella, Giorgio

    2016-03-01

    Human Parvovirus B19 (PVB19) is one of the most important pathogens that targets erythroid lineage. Many factors were mentioned for restriction to erythroid progenitor cells (EPCs). Previous studies showed that in non-permissive cells VP1 and VP2 (structural proteins) mRNAs were detected but could not translate to proteins. A bioinformatics study showed that this inhibition might be due to specific microRNAs (miRNAs) present in non-permissive cells but not in permissive EPCs. To confirm the hypothesis, we evaluated the effect of miRNAs on VP expression. CD34(+) HSCs were separated from cord blood. Then, CD34(+) cells were treated with differentiation medium to obtain CD36(+) EPCs. To evaluate the effect of miRNAs on VP expression in MCF7 and HEK-293 cell lines (non-permissive cells) and CD36(+) EPCs, dual luciferase assay was performed in presence of shRNAs against Dicer and Drosha to disrupt miRNA biogenesis. QRT-PCR was performed to check down-regulation of Dicer and Drosha after transfection. All measurements were done in triplicate. Data means were compared using one-way ANOVAs. MicroRNA prediction was done by the online microRNA prediction tools. No significant difference was shown in luciferase activity of CD36(+) EPCs after co-transfection with shRNAs, while it was significant in non-permissive cells. Our study revealed that miRNAs may be involved in inhibition of VP expression in non-permissive cells, although further studies are required to demonstrate which miRNAs exactly are involved in regulation of PVB19 replication. PMID:26878856

  8. Exacerbating Effects of Human Parvovirus B19 NS1 on Liver Fibrosis in NZB/W F1 Mice

    PubMed Central

    Hsu, Tsai-Ching; Tsai, Chun-Chou; Chiu, Chun-Ching; Hsu, Jeng-Dong; Tzang, Bor-Show

    2013-01-01

    Systemic lupus erythematosus (SLE) is an autoimmune disorder with unknown etiology that impacts various organs including liver. Recently, human parvovirus B19 (B19) is recognized to exacerbate SLE. However, the effects of B19 on liver in SLE are still unclear. Herein we aimed to investigate the effects of B19 on liver in NZB/W F1 mice by injecting subcutaneously with PBS, recombinant B19 NS1, VP1u or VP2, respectively. Our experimental results revealed that B19 NS1 protein significantly enhanced the TGF-β/Smad fibrotic signaling by increasing the expressions of TGF-β, Smad2/3, phosphorylated Smad2/3, Smad4 and Sp1. The consequent fibrosis-related proteins, PAI-1 and α-SMA, were also significantly induced in livers of NZB/W F1 mice receiving B19 NS1 protein. Accordingly, markedly increased collagen deposition was also observed in livers of NZB/W F1 mice receiving B19 NS1 protein. However, no significant difference was observed in livers of NZB/W F1 mice receiving B19 VP1u or VP2 as compared to the controls. These findings indicate that B19 NS1 plays a crucial role in exacerbating liver fibrosis in NZB/W F1 mice through enhancing the TGF-â/Smad fibrotic signaling. PMID:23840852

  9. VP1u phospholipase activity is critical for infectivity of full-length parvovirus B19 genomic clones.

    PubMed

    Filippone, Claudia; Zhi, Ning; Wong, Susan; Lu, Jun; Kajigaya, Sachiko; Gallinella, Giorgio; Kakkola, Laura; Söderlund-Venermo, Maria; Young, Neal S; Brown, Kevin E

    2008-05-10

    Three full-length genomic clones (pB19-M20, pB19-FL and pB19-HG1) of parvovirus B19 were produced in different laboratories. pB19-M20 was shown to produce infectious virus. To determine the differences in infectivity, all three plasmids were tested by transfection and infection assays. All three clones were similar in viral DNA replication, RNA transcription, and viral capsid protein production. However, only pB19-M20 and pB19-HG1 produced infectious virus. Comparison of viral sequences showed no significant differences in ITR or NS regions. In the capsid region, there was a nucleotide sequence difference conferring an amino acid substitution (E176K) in the phospholipase A2-like motif of the VP1-unique (VP1u) region. The recombinant VP1u with the E176K mutation had no catalytic activity as compared with the wild-type. When this mutation was introduced into pB19-M20, infectivity was significantly attenuated, confirming the critical role of this motif. Investigation of the original serum from which pB19-FL was cloned confirmed that the phospholipase mutation was present in the native B19 virus.

  10. Exacerbating effects of human parvovirus B19 NS1 on liver fibrosis in NZB/W F1 mice.

    PubMed

    Hsu, Tsai-Ching; Tsai, Chun-Chou; Chiu, Chun-Ching; Hsu, Jeng-Dong; Tzang, Bor-Show

    2013-01-01

    Systemic lupus erythematosus (SLE) is an autoimmune disorder with unknown etiology that impacts various organs including liver. Recently, human parvovirus B19 (B19) is recognized to exacerbate SLE. However, the effects of B19 on liver in SLE are still unclear. Herein we aimed to investigate the effects of B19 on liver in NZB/W F1 mice by injecting subcutaneously with PBS, recombinant B19 NS1, VP1u or VP2, respectively. Our experimental results revealed that B19 NS1 protein significantly enhanced the TGF-β/Smad fibrotic signaling by increasing the expressions of TGF-β, Smad2/3, phosphorylated Smad2/3, Smad4 and Sp1. The consequent fibrosis-related proteins, PAI-1 and α-SMA, were also significantly induced in livers of NZB/W F1 mice receiving B19 NS1 protein. Accordingly, markedly increased collagen deposition was also observed in livers of NZB/W F1 mice receiving B19 NS1 protein. However, no significant difference was observed in livers of NZB/W F1 mice receiving B19 VP1u or VP2 as compared to the controls. These findings indicate that B19 NS1 plays a crucial role in exacerbating liver fibrosis in NZB/W F1 mice through enhancing the TGF-â/Smad fibrotic signaling.

  11. VP1u phospholipase activity is critical for infectivity of full-length parvovirus B19 genomic clones✰

    PubMed Central

    Filippone, Claudia; Zhi, Ning; Wong, Susan; Lu, Jun; Kajigaya, Sachiko; Gallinella, Giorgio; Kakkola, Laura; Venermo, Maria S Söderlund; Young, Neal S.; Brown, Kevin E.

    2008-01-01

    Three full-length genomic clones (pB19-M20, pB19-FL and pB19-HG1) of parvovirus B19 were produced in different laboratories. pB19-M20 was shown to produce infectious virus. To determine the differences in infectivity, all three plasmids were tested by transfection and infection assays. All three clones were similar in viral DNA replication, RNA transcription, and viral capsid protein production. However, only pB19-M20 and pB19-HG1 produced infectious virus. Comparison of viral sequences showed no significant differences in ITR or NS regions. In the capsid region, there was a nucleotide sequence difference conferring an amino acid substitution (E176K) in the phospholipase A2-like motif of the VP1-unique (VP1u) region. The recombinant VP1u with the E176K mutation had no catalytic activity as compared with the wild-type. When this mutation was introduced into pB19-M20, infectivity was significantly attenuated, confirming the critical role of this motif. Investigation of the original serum from which pB19-FL was cloned confirmed that the phospholipase mutation was present in the native B19 virus. PMID:18252260

  12. Pure red cell aplasia caused by parvovirus B19 in a heart transplant recipient.

    PubMed

    Invernizzi, Rosangela; Bastia, Raffaella; Quaglia, Federica

    2016-09-01

    A case of parvovirus B19-induced pure red cell aplasia occurring in a heart transplant recipient is reported. The diagnosis of this rare but clinically important complication can be suspected on the basis of the pathognomonic morphological features of the bone marrow. PMID:27648265

  13. Strain- and Genotype-Specific Differences in Virulence of Paenibacillus larvae subsp. larvae, a Bacterial Pathogen Causing American Foulbrood Disease in Honeybees

    PubMed Central

    Genersch, Elke; Ashiralieva, Ainura; Fries, Ingemar

    2005-01-01

    Virulence variations of Paenibacillus larvae subsp. larvae, the causative agent of American foulbrood disease of honeybees, were investigated by analysis of 16 field isolates of this pathogen, belonging to three previously characterized genotypes, as well as the type strain (ATCC 9545) of P. larvae subsp. larvae, with exposure bioassays. We demonstrated that the strain-specific 50% lethal concentrations varied within an order of magnitude and that differences in amount of time for the pathogen to kill 100% of the infected hosts (LT100) correlated with genotype. One genotype killed rather quickly, with a mean LT100 of 7.8 ± 1.7 days postinfection, while the other genotypes acted more slowly, with mean LT100s of 11.2 ± 0.8 and 11.6 ± 0.6 days postinfection. PMID:16269801

  14. Capitalizing on tumor genotyping: Towards the design of mutation specific inhibitors of phosphoinsitide-3-kinase

    SciTech Connect

    Gabelli, Sandra B.; Duong-Ly, Krisna C.; Brower, Evan T.; Amzel, L. Mario

    2011-09-06

    PI3Ks catalyze the phosphorylation of the inositol hydroxyls of phosphoinositide membrane components. The changes in phosphorylation of the inositides recruit proteins to the plasma membrane that initiate important signaling cascades. PI3K{alpha}, one of the class IA PI3Ks, is highly mutated in cancers. All mutations analyzed result in an increase in enzymatic activity. The structures of this enzyme determined by X-ray diffraction, provide a framework for analyzing the possible structural effect of these mutations and their effect on the enzymatic activity. Many of the mutations occur at domain interfaces where they can affect domain interactions and relieve the inhibition of the wild-type enzyme by the nSH2 domain of p85. This mechanism is analogous to the mechanism of physiological activation by activated tyrosine-kinase receptors in which the phosphorylated tyrosine of the receptor (or their substrates) dislodges the nSH2 from its inhibitory position in the complex by competing with its binding to a loop in the helical domain. Other mutations in the kinase domain can directly affect the conformation of the catalytic site. One mutation, His1047Arg, uses a completely different mechanism: it changes the conformation of the C-terminal loop in such a way that it increases the interaction of the enzyme with the membrane, granting increased access to the phosphoinositide substrates. Taking advantage of the reliance of some cancers on the increased activity of mutated PI3K{alpha}, will require the development of isoform-specific, mutant-specific inhibitors. The structural, biochemical and physiological data that are becoming available for PI3Ks are an important first step in this direction.

  15. Gender-Specific Effect of Mthfr Genotype and Neonatal Vigabatrin Interaction on Synaptic Proteins in Mouse Cortex

    PubMed Central

    Blumkin, Elinor; Levav-Rabkin, Tamar; Melamed, Osnat; Galron, Dalia; Golan, Hava M

    2011-01-01

    The enzyme methylenetetrahydrofolate reductase (MTHFR) is a part of the homocysteine and folate metabolic pathways, affecting the methylations of DNA, RNA, and proteins. Mthfr deficiency was reported as a risk factor for neurodevelopmental disorders such as autism spectrum disorder and schizophrenia. Neonatal disruption of the GABAergic system is also associated with behavioral outcomes. The interaction between the epigenetic influence of Mthfr deficiency and neonatal exposure to the GABA potentiating drug vigabatrin (GVG) in mice has been shown to have gender-dependent effects on mice anxiety and to have memory impairment effects in a gender-independent manner. Here we show that Mthfr deficiency interacts with neonatal GABA potentiation to alter social behavior in female, but not male, mice. This impairment was associated with a gender-dependent enhancement of proteins implicated in excitatory synapse plasticity in the female cortex. Reelin and fragile X mental retardation 1 protein (FMRP) levels and membrane GluR1/GluR2 ratios were elevated in wild-type mice treated neonatally with GVG and in Mthfr+/− mice treated with saline, but not in Mthfr+/− mice treated with GVG, compared with control groups (wild type treated with saline). A minor influence on the levels of these proteins was observed in male mice cortices, possibly due to high basal protein levels. Interaction between gender, genotype, and treatment was also observed in the GABA pathway. In female mice, GABA Aα2/gephyrin ratios were suppressed in all test groups; in male mice, a genotype-specific enhancement of GABA Aα2/gephyrin was observed. The lack of an effect on either reln or Fmr1 transcription suggests post-transcriptional regulation of these genes. Taken together, these findings suggest that Mthfr deficiency may interact with neonatal GABA potentiation in a gender-dependent manner to interrupt synaptic function. This may illustrate a possible mechanism for the epigenetic involvement of Mthfr

  16. Human adenovirus 2 E1B-19K and E1B-53K tumor antigens: antipeptide antibodies targeted to the NH2 and COOH termini.

    PubMed Central

    Green, M; Brackmann, K H; Lucher, L A; Symington, J S; Kramer, T A

    1983-01-01

    The human adenovirus 2 (Ad2) transforming region is located in the left 11.1% of the viral genome and encodes two early transcription units, E1A and E1B. Based on the amino acid sequence deduced from the Ad2 E1B DNA sequence (Gingeras et al., J. Biol. Chem. 257:13475-13491, 1982), we have prepared antibodies against synthetic peptides, 8 to 16 amino acids in length, encoded at the NH2 and COOH termini of the major E1B-19K and E1B-53K tumor antigens. The antipeptide antibodies immunoprecipitated the targeted E1B-19K or E1B-53K tumor antigens from extracts of Ad2-infected cells. The specificity of the peptide competition studies. Antipeptide antibodies directed to the NH2 and COOH termini immunoprecipitated the E1B-19K and E1B-53K tumor antigens from two Ad2-transformed rat cell lines, F17 and F4, providing evidence that identical tumor antigens are synthesized in Ad2-infected and Ad2-transformed cells. These results show that the E1B-19K and E1B-53K T antigens are not processed proteolytically at either the NH2 or COOH terminus. Our data provide strong evidence at the protein level that the E1B-19K and E1B-53K tumor antigens partially overlap in DNA sequence, with the E1B-19K initiating translation at the first ATG at nucleotide 1711 in translation reading frame 1 and the E1B-53K tumor antigen initiating translation at the second ATG at nucleotide 2016 in reading frame 3. This confirms the results of others on the N-terminal amino acid sequence of E1B-19K and theoretical deductions based on the DNA sequence. Our findings prove that the large E1B-53K T antigen initiates translation at the second ATG at nucleotide 2016 and not at equally plausible initiation codons located farther downstream at nucleotides 2202 and 2235. Thus, the E1B-53K T antigen is another example of a protein which initiates translation at an internal ATG rather than at the 5'-proximal ATG. Images PMID:6632083

  17. A protected area influences genotype-specific survival and the structure of a Canis hybrid zone.

    PubMed

    Benson, John F; Patterson, Brent R; Mahoney, Peter J

    2014-02-01

    It is widely recognized that protected areas can strongly influence ecological systems and that hybridization is an important conservation issue. However, previous studies have not explicitly considered the influence of protected areas on hybridization dynamics. Eastern wolves are a species of special concern and their distribution is largely restricted to a protected population in Algonquin Provincial Park (APP), Ontario, Canada, where they are the numerically dominant canid. We studied intrinsic and extrinsic factors influencing survival and cause-specific mortality of hybrid and parental canids in the three-species hybrid zone between eastern wolves, eastern coyotes, and gray wolves in and adjacent to APP. Mortality risk for eastern wolves in areas adjacent to APP was significantly higher than for other sympatric Canis types outside of APP, and for eastern wolves and other canids within APP. Outside of APP, the annual mortality rate of all canids by harvest (24%) was higher than for other causes of death (4-7%). Furthermore, eastern wolves (hazard ratio = 3.5) and nonresidents (transients and dispersing animals, hazard ratio = 2.7) were more likely to die from harvest relative to other Canis types and residents, respectively. Thus, eastern wolves dispersing from APP were especially vulnerable to harvest mortality. For residents, eastern wolf survival was more negatively influenced by increased road density than for other Canis types, further highlighting the sensitivity of eastern wolves to human disturbance. A cycle of dispersal from APP followed by high rates of mortality and hybridization appears to maintain eastern wolves at low density adjacent to APP, limiting the potential for expansion beyond the protected area. However, high survival and numerical dominance of eastern wolves within APP suggest that protected areas can allow rare hybridizing species to persist even if their demographic performance is compromised and barriers to hybridization are largely

  18. Effector-Triggered Immunity Determines Host Genotype-Specific Incompatibility in Legume-Rhizobium Symbiosis.

    PubMed

    Yasuda, Michiko; Miwa, Hiroki; Masuda, Sachiko; Takebayashi, Yumiko; Sakakibara, Hitoshi; Okazaki, Shin

    2016-08-01

    Symbiosis between legumes and rhizobia leads to the formation of N2-fixing root nodules. In soybean, several host genes, referred to as Rj genes, control nodulation. Soybean cultivars carrying the Rj4 gene restrict nodulation by specific rhizobia such as Bradyrhizobium elkanii We previously reported that the restriction of nodulation was caused by B. elkanii possessing a functional type III secretion system (T3SS), which is known for its delivery of virulence factors by pathogenic bacteria. In the present study, we investigated the molecular basis for the T3SS-dependent nodulation restriction in Rj4 soybean. Inoculation tests revealed that soybean cultivar BARC-2 (Rj4/Rj4) restricted nodulation by B. elkanii USDA61, whereas its nearly isogenic line BARC-3 (rj4/rj4) formed nitrogen-fixing nodules with the same strain. Root-hair curling and infection threads were not observed in the roots of BARC-2 inoculated with USDA61, indicating that Rj4 blocked B. elkanii infection in the early stages. Accumulation of H2O2 and salicylic acid (SA) was observed in the roots of BARC-2 inoculated with USDA61. Transcriptome analyses revealed that inoculation of USDA61, but not its T3SS mutant in BARC-2, induced defense-related genes, including those coding for hypersensitive-induced responsive protein, which act in effector-triggered immunity (ETI) in Arabidopsis. These findings suggest that B. elkanii T3SS triggers the SA-mediated ETI-type response in Rj4 soybean, which consequently blocks symbiotic interactions. This study revealed a common molecular mechanism underlying both plant-pathogen and plant-symbiont interactions, and suggests that establishment of a root nodule symbiosis requires the evasion or suppression of plant immune responses triggered by rhizobial effectors. PMID:27373538

  19. Human Parvovirus B19 NS1 Protein Aggravates Liver Injury in NZB/W F1 Mice

    PubMed Central

    Tsai, Chun-Chou; Chiu, Chun-Ching; Hsu, Jeng-Dong; Hsu, Huai-Sheng; Tzang, Bor-Show; Hsu, Tsai-Ching

    2013-01-01

    Human parvovirus B19 (B19) has been associated with a variety of diseases. However, the influence of B19 viral proteins on hepatic injury in SLE is still obscure. To elucidate the effects of B19 viral proteins on livers in SLE, recombinant B19 NS1, VP1u or VP2 proteins were injected subcutaneously into NZB/W F1 mice, respectively. Significant expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected in NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Markedly hepatocyte disarray and lymphocyte infiltration were observed in livers from NZB/WF 1 mice receiving B19 NS1 as compared to those mice receiving PBS. Additionally, significant increases of Tumor Necrosis Factor –α (TNF-α), TNF-α receptor, IκB kinase –α (IKK-α), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IκB) and nuclear factor-kappa B (NF-κB) were detected in livers from NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Accordingly, significant increases of matrix metalloproteinase-9 (MMP9) and U-plasminogen activator (uPA) were also detected in livers from NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Contrarily, no significant variation on livers from NZB/W F1 mice receiving B19 VP1u or VP2 was observed as compared to those mice receiving PBS. These findings firstly demonstrated the aggravated effects of B19 NS1 but not VP1u or VP2 protein on hepatic injury and provide a clue in understanding the role of B19 NS1 on hepatic injury in SLE. PMID:23555760

  20. Human parvovirus B19 VP1u Protein as inflammatory mediators induces liver injury in naïve mice.

    PubMed

    Hsu, Tsai-Ching; Chiu, Chun-Ching; Chang, Shun-Chih; Chan, Hsu-Chin; Shi, Ya-Fang; Chen, Tzy-Yen; Tzang, Bor-Show

    2016-01-01

    Human parvovirus B19 (B19V) is a human pathogen known to be associated with many non-erythroid diseases, including hepatitis. Although B19V VP1-unique region (B19-VP1u) has crucial roles in the pathogenesis of B19V infection, the influence of B19-VP1u proteins on hepatic injury is still obscure. This study investigated the effect and possible inflammatory signaling of B19-VP1u in livers from BALB/c mice that were subcutaneously inoculated with VP1u-expressing COS-7 cells. The in vivo effects of B19-VP1u were analyzed by using live animal imaging system (IVIS), Haematoxylin-Eosin staining, gel zymography, and immunoblotting after inoculation. Markedly hepatocyte disarray and lymphocyte infiltration, enhanced matrix metalloproteinase (MMP)-9 activity and increased phosphorylation of p38, ERK, IKK-α, IκB and NF-κB (p-p65) proteins were observed in livers from BALB/c mice receiving COS-7 cells expressing B19-VP1u as well as the significantly increased CRP, IL-1β and IL-6. Notably, IFN-γ and phosphorylated STAT1, but not STAT3, were also significantly increased in the livers of BALB/c mice that were subcutaneously inoculated with VP1u-expressing COS-7 cells. These findings revealed the effects of B19-VP1u on liver injury and suggested that B19-VP1u may have a role as mediators of inflammation in B19V infection.

  1. Human parvovirus B19 NS1 protein aggravates liver injury in NZB/W F1 mice.

    PubMed

    Tsai, Chun-Chou; Chiu, Chun-Ching; Hsu, Jeng-Dong; Hsu, Huai-Sheng; Tzang, Bor-Show; Hsu, Tsai-Ching

    2013-01-01

    Human parvovirus B19 (B19) has been associated with a variety of diseases. However, the influence of B19 viral proteins on hepatic injury in SLE is still obscure. To elucidate the effects of B19 viral proteins on livers in SLE, recombinant B19 NS1, VP1u or VP2 proteins were injected subcutaneously into NZB/W F1 mice, respectively. Significant expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected in NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Markedly hepatocyte disarray and lymphocyte infiltration were observed in livers from NZB/WF 1 mice receiving B19 NS1 as compared to those mice receiving PBS. Additionally, significant increases of Tumor Necrosis Factor -α (TNF-α), TNF-α receptor, IκB kinase -α (IKK-α), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IκB) and nuclear factor-kappa B (NF-κB) were detected in livers from NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Accordingly, significant increases of matrix metalloproteinase-9 (MMP9) and U-plasminogen activator (uPA) were also detected in livers from NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Contrarily, no significant variation on livers from NZB/W F1 mice receiving B19 VP1u or VP2 was observed as compared to those mice receiving PBS. These findings firstly demonstrated the aggravated effects of B19 NS1 but not VP1u or VP2 protein on hepatic injury and provide a clue in understanding the role of B19 NS1 on hepatic injury in SLE.

  2. Development of a Melting Curve-Based Allele-Specific PCR of Apolipoprotein E (APOE) Genotyping Method for Genomic DNA, Guthrie Blood Spot, and Whole Blood.

    PubMed

    Chen, Chia-Hsiang

    2016-01-01

    Genetic polymorphisms of apolipoprotein E (APOE) are associated with various health conditions and diseases, such as Alzheimer's disease, cardiovascular diseases, type 2 diabetes, etc. Hence, genotyping of APOE has broad applications in biomedical research and clinical settings, particularly in the era of precision medicine. The study aimed to develop a convenient and accurate method with flexible throughput to genotype the APOE polymorphisms. A melting curve-based allele-specific PCR method was developed to genotype two single nucleotide polymorphisms (SNPs) of APOE, i.e. rs429358 at codon 112 and rs7412 at codon 158. These two SNPs determine the genotype of APOE2, E3, and E4. PCR-based Sanger sequencing was used as the reference method for APOE genotyping. A 100% concordance rate was obtained in 300 subjects between the melting curve-based allele-specific PCR method and the Sanger sequencing method. This method was applied to a genetic association analysis of APOE and schizophrenia consisting of 711 patients with schizophrenia and 665 control subjects from Taiwan. However, no significant differences in the allele and genotype frequencies were detected between these two groups. Further experiments showed that DNA dissolved from blood collected on Guthrie filter paper and total blood cell lysate without DNA extraction can be used in the melting curve-based allele-specific PCR method. Thus, we suggest that this is a fast, accurate and robust APOE genotyping method with a flexible throughput and suitable for DNA template from different preparations. This convenient method shall meet the different needs of various research and clinical laboratories. PMID:27078154

  3. Strain specific genotype-environment interactions and evolutionary potential for body mass in brook charr (Salvelinus fontinalis).

    PubMed

    Crespel, Amélie; Bernatchez, Louis; Audet, Céline; Garant, Dany

    2013-03-01

    Discriminating between genetic and environmental causes of phenotypic variation is an essential requirement for understanding the evolutionary potential of populations. However, the extent to which genetic variation differs among conspecific groups and environments during ontogeny has rarely been investigated. In this study, the genetic basis of body mass was measured in three divergent strains of brook charr (Salvelinus fontinalis) in different rearing environments and at different time periods. The results indicate that body mass was a heritable trait in all strains but that the level of heritability greatly differed among strains. Moreover, heritability estimates of each strain varied differently according to environmental rearing conditions, and cross-environments correlations were all significantly lower than unity, indicating strain-specific patterns of genotype-environment interactions. Heritability estimates also varied throughout ontogeny and decreased by 50% from 9 to 21 months of age. This study highlights the divergence in genetic architecture and evolutionary potential among these strains and emphasizes the importance of considering the strain-specific potential of the response to selection according to environmental variation.

  4. Th17-Related Cytokines in Systemic Lupus Erythematosus Patients with Dilated Cardiomyopathies: A Possible Linkage to Parvovirus B19 Infection

    PubMed Central

    Chen, Der-Yuan; Chen, Yi-Ming; Lan, Joung-Liang

    2014-01-01

    Dilated cardiomyopathies (DCM) are a major cause of mortality in patients with systemic lupus erythematosus (SLE). Immune responses induced by human parvovirus B19 (B19) are considered an important pathogenic mechanism in myocarditis or DCM. However, little is known about Th17-related cytokines in SLE patients with DCM about the linkage with B19 infection. IgM and IgG against B19 viral protein, and serum levels of Th17-related cytokines were determined using ELISA in eight SLE patients with DCM and six patients with valvular heart disease (VHD). Humoral responses of anti-B19-VP1u and anti-B19-NS1 antibody were assessed using Western blot and B19 DNA was detected by nested Polymerase Chain Reaction (PCR). Levels of interleukin (IL)-17, IL-6, IL-1β, and tumor necrosis factor (TNF)-α were significantly higher in SLE patients with DCM (mean ± SEM, 390.99±125.48 pg/ml, 370.24±114.09 pg/ml, 36.01±16.90 pg/ml, and 183.84±82.94 pg/ml, respectively) compared to healthy controls (51.32±3.04 pg/ml, p<0.001; 36.88±6.64 pg/ml, p<0.001; 5.39±0.62 pg/ml, p<0.005; and 82.13±2.42 pg/ml, p<0.005, respectively). Levels of IL-17 and IL-6 were higher in SLE patients with DCM versus those with VHD (both p<0.01). Five (62.5%) of DCM patients had detectable anti-B19-NS1 IgG and four (50.0%) of them had anti-B19-VP1u IgG, whereas only one (16.7%) of VHD patients had detectable anti-B19-NS1 IgG and anti-B19-VP1u IgG. Serum levels of IL-17, IL-6 and IL-1β were markedly higher in SLE patients with anti-B19-VP1u IgG and anti-B19-NS1 IgG compared to those without anti-B19-VP1u IgG or anti-B19-NS1 IgG, respectively. These suggest a potential association of B19 with DCM and Th17-related cytokines implicated in the pathogenesis of DCM in SLE patients. PMID:25462010

  5. Th17-related cytokines in systemic lupus erythematosus patients with dilated cardiomyopathies: a possible linkage to parvovirus B19 infection.

    PubMed

    Chen, Der-Yuan; Chen, Yi-Ming; Tzang, Bor-Show; Lan, Joung-Liang; Hsu, Tsai-Ching

    2014-01-01

    Dilated cardiomyopathies (DCM) are a major cause of mortality in patients with systemic lupus erythematosus (SLE). Immune responses induced by human parvovirus B19 (B19) are considered an important pathogenic mechanism in myocarditis or DCM. However, little is known about Th17-related cytokines in SLE patients with DCM about the linkage with B19 infection. IgM and IgG against B19 viral protein, and serum levels of Th17-related cytokines were determined using ELISA in eight SLE patients with DCM and six patients with valvular heart disease (VHD). Humoral responses of anti-B19-VP1u and anti-B19-NS1 antibody were assessed using Western blot and B19 DNA was detected by nested Polymerase Chain Reaction (PCR). Levels of interleukin (IL)-17, IL-6, IL-1β, and tumor necrosis factor (TNF)-α were significantly higher in SLE patients with DCM (mean ± SEM, 390.99±125.48 pg/ml, 370.24±114.09 pg/ml, 36.01±16.90 pg/ml, and 183.84±82.94 pg/ml, respectively) compared to healthy controls (51.32±3.04 pg/ml, p<0.001; 36.88±6.64 pg/ml, p<0.001; 5.39±0.62 pg/ml, p<0.005; and 82.13±2.42 pg/ml, p<0.005, respectively). Levels of IL-17 and IL-6 were higher in SLE patients with DCM versus those with VHD (both p<0.01). Five (62.5%) of DCM patients had detectable anti-B19-NS1 IgG and four (50.0%) of them had anti-B19-VP1u IgG, whereas only one (16.7%) of VHD patients had detectable anti-B19-NS1 IgG and anti-B19-VP1u IgG. Serum levels of IL-17, IL-6 and IL-1β were markedly higher in SLE patients with anti-B19-VP1u IgG and anti-B19-NS1 IgG compared to those without anti-B19-VP1u IgG or anti-B19-NS1 IgG, respectively. These suggest a potential association of B19 with DCM and Th17-related cytokines implicated in the pathogenesis of DCM in SLE patients.

  6. Development of a high-density intra-specific linkage map of lettuce using genotyping by sequencing (GBS)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genotyping by sequencing (GBS) has been developed as an affordable application of next-generation sequencing for the purposes of discovering and genotyping SNPs in a variety of crop species and populations. In this study we employed a double restriction enzyme digestion protocol (HindIII and NlaIII)...

  7. [Rh-D genotyping for exon 2, 5 and 7 of German and Japanese blood donors with sequence specific polymerase chain reaction].

    PubMed

    Maas, J H; Legler, T J; Lynen, R; Blaschke, V; Ohto, H; Köhler, M

    1997-01-01

    RHD genotyping from fetal cells was applied for the detection of the RHD gene in the fetus of immunized Rh-D-negative women. Additionally, RHD genotyping was applied for the characterization of Rh-D variants. Although 44 nucleotide substitutions are known to code for 35 amino acid differences between the RHCE and the RHD gene, only a few polymorphisms have been investigated yet. We investigated 7 RHD-specific nucleotides on exons 2, 5, and 7 with sequence-specific primers and 1 nucleotide with ligation-based typing. All RHD genotyping results were correlated with serological results and established genotyping methods in 116 German and 98 Japanese blood donors, because different genetic sequences coding for Rh-D polypeptides have been described in different ethnic groups. Sequence-specific amplification of D-specific sequences was concordant with the serological result in all blood donors tested. However, ligation-based typing on exon 5 gave false-negative results in 7 donors. In summary, 5 new sequence-specific PCRs have been evaluated for further characterization of Rh-D variants. Furthermore, the methods described allow nested PCR and thus may help in determination of the fetal RhD status from maternal peripheral blood during pregnancy.

  8. Genotypic and phenotypic characterization of Lactobacillus casei strains isolated from different ecological niches suggests frequent recombination and niche specificity.

    PubMed

    Cai, Hui; Rodríguez, Beatriz T; Zhang, Wei; Broadbent, Jeff R; Steele, James L

    2007-08-01

    Lactobacillus casei strains are lactic acid bacteria (LAB) that colonize diverse ecological niches, and have broad commercial applications. To probe their evolution and phylogeny, 40 L. casei strains were characterized; the strains included isolates from plant materials (n=9), human gastrointestinal tracts (n=7), human blood (n=1), cheeses from different geographical locations (n=22), and one strain of unknown origin. API biochemical testing identified niche-specific carbohydrate fermentation profiles. A multilocus sequence typing (MLST) scheme was developed for L. casei. Partial sequencing of six housekeeping genes (ftsZ, metRS, mutL, nrdD, pgm and polA) revealed between 11 (nrdD) and 20 (mutL) allelic types, as well as 36 sequence types. Phylogenetic analysis of MLST data by Reticulate and split decomposition analysis indicated frequent intra-species recombination. Purifying selection was detected, and is likely to have contributed to the evolution of certain L. casei genes. Pulsed-field gel electrophoresis (PFGE) using SfiI was able to discriminate all the isolates, even those not differentiated by MLST. Phylogenetic trees reconstructed based on the MLST data using minimum evolution algorithm, and the SfiI-PFGE restriction patterns using the unweighted-pair group method with arithmetic mean (UPGMA), revealed consensus clusters of strains specific to cheese and silage. Topological discrepancies between the MLST and PFGE trees were also observed, suggesting that intragenic point mutations have accumulated at a slower rate than indels and genome rearrangements in L. casei. The L. casei population analysed in this study demonstrated both a high level of phenotypic and genotypic diversity, as well as specificity to different ecological niches. PMID:17660430

  9. Quantitative direct probe method for the detection of parvovirus B19.

    PubMed

    Boggino, H; Payne, D A

    2000-01-01

    Parvovirus B19 infection is associated with anemia and spontaneous abortions. While many qualitative assays are available, a few molecular-based quantitative methods have been described. This study reports the development and optimization of a quantitative direct-probe method for the detection of Parvovirus B19 DNA. Different concentrations of RNA probes were used to identify the optimal conditions for hybridizing to the target DNA. Detection of DNA was linear between concentrations of 2 ng/ml to 200 pg/ml. Because this method requires no enzymatic amplification, it is not susceptible to amplifier contamination or enzymatic inhibitors, and it can be applied to serum samples or paraffin-embedded tissue.

  10. Quantitative direct probe method for the detection of parvovirus B19.

    PubMed

    Boggino, H; Payne, D A

    2000-01-01

    Parvovirus B19 infection is associated with anemia and spontaneous abortions. While many qualitative assays are available, a few molecular-based quantitative methods have been described. This study reports the development and optimization of a quantitative direct-probe method for the detection of Parvovirus B19 DNA. Different concentrations of RNA probes were used to identify the optimal conditions for hybridizing to the target DNA. Detection of DNA was linear between concentrations of 2 ng/ml to 200 pg/ml. Because this method requires no enzymatic amplification, it is not susceptible to amplifier contamination or enzymatic inhibitors, and it can be applied to serum samples or paraffin-embedded tissue. PMID:10645984

  11. Organ-specific phosphorus-allocation patterns and transcript profiles linked to phosphorus efficiency in two contrasting wheat genotypes.

    PubMed

    Aziz, Tariq; Finnegan, Patrick M; Lambers, Hans; Jost, Ricarda

    2014-04-01

    Recent studies have identified genotypic variation in phosphorus (P) efficiency, but rarely have the underlying mechanisms been described at the molecular level. We demonstrate that the highly P-efficient wheat (Triticum aestivum L.) cultivar Chinese 80-55 maintains higher inorganic phosphate (Pi ) concentrations in all organs upon Pi withdrawal in combination with higher Pi acquisition in the presence of Pi when compared with the less-efficient cultivar Machete. These findings correlated with differential organ-specific expression of Pi transporters TaPHT1;2, TaPHT1;5, TaPHT1;8, TaPHT2;1 and H(+) -ATPase TaHa1. Observed transcript level differences between the cultivars suggest that higher de novo phospholipid biosynthetic activities in Pi -limited elongating basal leaf sections are another crucial adaptation in Chinese 80-55 for sustaining growth upon Pi withdrawal. These activities may be supported through enhanced breakdown of starch in Chinese 80-55 stems as suggested by higher TaGPho1 transcript levels. Chinese 80-55 fine roots on the other hand show strong suppression of transcripts involved in glycolysis, transcriptional regulation and ribosomal activities. Our work reveals major differences in the way the two contrasting cultivars allocate Pi and organic P compounds between source and sink tissues and in the acclimation of their metabolism to changes in Pi availability.

  12. Pneumocystis jiroveci in Portuguese immunocompromised patients: association of specific ITS genotypes with treatment failure, bad clinical outcome and childhood.

    PubMed

    Matos, Olga; Lee, Chao-Hung; Jin, Shaoling; Li, Baozheng; Costa, Marina C; Gonçalves, Luzia; Antunes, Francisco

    2003-11-01

    We analyzed the genetic variation among isolates of Pneumocystis jiroveci from Portuguese immunocompromised patients with PCP at the internal transcribed spacer (ITS) regions of the nuclear rRNA operon and at the dihydropteroate synthase (DHPS) gene. Pulmonary secretions from 42 patients with PCP corresponding to 43 episodes were studied. Demographic, immunological, and clinical data were obtained from all patients. By combining the two regions ITS1 and ITS2, we found 17 different ITS types of P. jiroveci, two of them were new types (Pb and Pe). The four most prevalent ITS types were Eg (23.3%), Eb and Ne (11.6% each), and Bi (9.3%). A single type was detected in 95.3% of the samples and 4.7% had mixed infections with three different ITS types. DHPS mutants were present in 17 (46%), and the wildtype was present in 20 (54%) of 37 isolates. No association was found between ITS and DHPS types and between DHPS types and therapy or response to anti-PCP treatment. Type Ne presented an association with negative response to anti-PCP treatment (P<0.001) and with death before 120 days after PCP diagnosis (P=0.025). Type Eb was significantly more common in children than in adults (P=0.001). Our data suggest an association of specific ITS genotypes with treatment failure, bad clinical outcome and childhood.

  13. Excretion of Brucella abortus vaccine B19 strain during a reproductive cycle in dairy cows

    PubMed Central

    Pacheco, W. A.; Genovez, M. E.; Pozzi, C. R.; Silva, L. M. P.; Azevedo, S. S.; Did, C. C.; Piatti, R. M.; Pinheiro, E. S.; Castro, V.; Miyashiro, S.; Gambarini, M. L.

    2012-01-01

    This paper aimed to determine the excretion period of B19 vaccine strain during a complete reproductive cycle (from estrus synchronization, artificial insemination, pregnancy and until 30 days after parturition) of dairy cows from 3 to 9 years old that were previously vaccinated from 3 to 8 months. Three groups were monitored with monthly milk and urine collection during 12 months: G1 with seven cows from 3 to 4 years old; G2 with three cows from 5 to 6 years old; and G3 with four cows from 7 to 9 years old. Urine and milk samples were submitted to bacteriological culture and urine and PCR reactions for detection of Brucella spp. and PCR-multiplex for B19 strain identification. Ring test (RT) was also performed in the milk samples, and serum samples were tested by buffered acidified plate antigen test (BAPA). All animals were serologically negative at BAPA and Brucella spp. was not isolated from both urine and milk samples. RT revealed 13/210 (6.2%) positive milk samples. PCR reactions detected DNA of Brucella spp. in 86/420 (20.5%) samples. In urine it was found a significantly higher frequency (35.2%; 74/210) than in milk (5.7%; 12/210), more frequently from the estrus to 150 days of pregnancy and after parturition (6.7%; 10/150), and from 150 days of pregnancy to parturition (3.4%; 2/60), and they were all identified as B19 strain. In three groups, intermittent excretion of B19 strain was detected mainly in urine samples, which confirmed its multiplication and persistence in cows for until 9 years. PMID:24031869

  14. Genetic Diversity within Human Erythroviruses: Identification of Three Genotypes

    PubMed Central

    Servant, Annabelle; Laperche, Syria; Lallemand, Francis; Marinho, Valérie; De Saint Maur, Guillemette; Meritet, Jean François; Garbarg-Chenon, Antoine

    2002-01-01

    B19 virus is a human virus belonging to the genus Erythrovirus. The genetic diversity among B19 virus isolates has been reported to be very low, with less than 2% nucleotide divergence in the whole genome sequence. We have previously reported the isolation of a human erythrovirus isolate, termed V9, whose sequence was markedly distinct (>11% nucleotide divergence) from that of B19 virus. To date, the V9 isolate remains the unique representative of a new variant in the genus Erythrovirus, and its taxonomic position is unclear. We report here the isolation of 11 V9-related viruses. A prospective study conducted in France between 1999 and 2001 indicates that V9-related viruses actually circulate at a significant frequency (11.4%) along with B19 viruses. Analysis of the nearly full-length genome sequence of one V9-related isolate (D91.1) indicates that the D91.1 sequence clusters together with but is notably distant from the V9 sequence (5.3% divergence) and is distantly related to B19 virus sequences (13.8 to 14.2% divergence). Additional phylogenetic analysis of partial sequences from the V9-related isolates combined with erythrovirus sequences available in GenBank indicates that the erythrovirus group is more diverse than thought previously and can be divided into three well-individualized genotypes, with B19 viruses corresponding to genotype 1 and V9-related viruses being distributed into genotypes 2 and 3. PMID:12186896

  15. Visualization of the Externalized VP2 N Termini of Infectious Human Parvovirus B19

    PubMed Central

    Kaufmann, Bärbel; Chipman, Paul R.; Kostyuchenko, Victor A.; Modrow, Susanne; Rossmann, Michael G.

    2008-01-01

    The structures of infectious human parvovirus B19 and empty wild-type particles were determined by cryoelectron microscopy (cryoEM) to 7.5-Å and 11.3-Å resolution, respectively, assuming icosahedral symmetry. Both of these, DNA filled and empty, wild-type particles contain a few copies of the minor capsid protein VP1. Comparison of wild-type B19 with the crystal structure and cryoEM reconstruction of recombinant B19 particles consisting of only the major capsid protein VP2 showed structural differences in the vicinity of the icosahedral fivefold axes. Although the unique N-terminal region of VP1 could not be visualized in the icosahedrally averaged maps, the N terminus of VP2 was shown to be exposed on the viral surface adjacent to the fivefold β-cylinder. The conserved glycine-rich region is positioned between two neighboring, fivefold-symmetrically related VP subunits and not in the fivefold channel as observed for other parvoviruses. PMID:18508892

  16. Autoimmune Hemolytic Anemia Triggered by Infection with Human Parvovirus B19 after Total Abdominal Colectomy for Ulcerative Colitis.

    PubMed

    Iida, Tomoya; Satoh, Shuji; Nakagaki, Suguru; Shimizu, Haruo; Kaneto, Hiroyuki

    2016-01-01

    A 50-year-old man was admitted to our hospital for an adhesive ileus 14 years after total abdominal colectomy for ulcerative colitis (UC). The ileus decreased with conservative treatment, however, autoimmune hemolytic anemia (AIHA) was diagnosed due to worsening anemia, a positive direct Coombs test, low haptoglobin, high lactase dehydrogenase, reticulocytosis, and an increase in the erythroblastic series in a bone-marrow examination. Human parvovirus B19 (PV-B19) IgM and PV-B19 DNA were present, indicating the development of AIHA triggered by an infection with PV-B19. The patient is currently being monitored after spontaneous remission. This is the first report of UC after total abdominal colectomy complicated by AIHA triggered by PV-B19 infection.

  17. The gender-specific apolipoprotein E genotype influence on the distribution of plasma lipids and apolipoproteins in the population of Rochester, Minnesota. II. Regression relationships with concomitants

    SciTech Connect

    Reilly, S.L.; Sing, C.F. ); Ferrell, R.E. ); Kottke, B.A. )

    1992-12-01

    The influence of the apolipoprotein E (Apo E) polymorphism and gender on the regression relationships between each of nine plasma lipid and apolipoprotein traits (total cholesterol; ln triglycerides; high-density-lipoprotein chloesterol; apolipoproteins AI, AII, B, and CII; ln CIII; and ln E) and four concomitants (age, weight, waist-to-hip ratio, and smoking) was studied in 507 unrelated individuals representative of the adult population of Rochester, MN. Analyses are presented separately for females and males. Each lipid and apolipoprotein trait exhibited at least one Apo E genotype-specific regression relationship with the concomitants investigated in this study. In most cases the heterogeneity of regression was associated with differences between the [var epsilon]32 and [var epsilon]33 genotype. This study documents that the influence of Apo E genotype on average levels of plasma lipids and apolipoproteins varies among subdivisions of the population defined by age, body size, and smoking status. 61 refs., 1 fig., 5 tabs.

  18. Systemic, genotype-specific induction of two herbivore-deterrent iridoid glycosides in Plantago lanceolata L. in response to fungal infection by Diaporthe adunca (Rob.) Niessel.

    PubMed

    Marak, Hamida B; Biere, Arjen; Van Damme, Jos M M

    2002-12-01

    Iridoid glycosides are a group of terpenoid secondary plant compounds known to deter generalist insect herbivores. In ribwort plantain (Plantago lanceolata), the iridoid glycosides aucubin and catalpol can be induced following damage by insect herbivores. In this study, we investigated whether the same compounds can be induced following infection by the fungal pathogen Diaporthe adunca, the causal agent of a stalk disease in P. lanceolata. Significant induction of aucubin and catalpol was observed in two of the three plant genotypes used in this study following inoculation with the pathogen. In one of the genotypes, induction occurred within 6 hr after inoculation, and no decay was observed within 8 days. The highest level of induction was observed in reproductive tissues (spikes and stalks) where infection took place. In these tissues, iridoid glycoside levels in infected plants were, on average, 97% and 37% higher than the constitutive levels in the corresponding control plants, respectively. Significant induction was also observed in leaves (24%) and roots (17%). In addition to significant genotypic variation in the level of induction, we found genetic variation for the tissue-specific pattern of induction, further broadening the scope for evolutionary fine-tuning of induced responses. Recent studies have revealed a negative association between iridoid glycoside levels in P. lanceolata genotypes and the amount of growth and reproduction of D. adunca that these genotypes support. However, for the three genotypes used in the present study, differences in resistance were not related to their constitutive or induced levels of iridoid glycosides, suggesting that additional resistance mechanisms are important in this host-pathogen system. We conclude that iridoid glycosides in P. lanceolata can be induced both by arthropods and pathogenic micro-organisms. Pathogen infection could, therefore, potentially enhance resistance to generalist insect herbivores in this

  19. Type-specific detection of human papillomaviruses in Kazakh esophageal squamous cell carcinoma by genotyping both E6 and L1 genes with MALDI-TOF mass spectrometry

    PubMed Central

    Dong, Hong-Chao; Cui, Xiao-Bin; Wang, Liang-Hai; Li, Man; Shen, Yao-Yuan; Zhu, Jian-Bo; Li, Cheng-Fang; Hu, Jian-Ming; Li, Shu-Gang; Yang, Lei; Zhang, Wen-Jie; Chen, Yun-Zhao; Li, Feng

    2015-01-01

    Background: Many studies have suggested a relationship between human papillomavirus (HPV) infection and the risk of esophageal squamous cell carcinoma (ESCC). However, findings are inconclusive, potentially because of geographic heterogeneity and variations in detection methods. Objectives: We sought to further investigate the prevalence of HPV with a new detection method, the MassARRAY Sequenom technique, in esophageal squamous cell carcinomas occurring in patients belonging to Kazakh populations in Xinjiang, China. Study design: In the present study, a novel genotyping method for detecting 30 HPV genotypes, specifically by genotyping both the HPV E6 and L1 genes with multiplex PCR using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (PCR-MS) was first adopted to evaluate HPV genotypes in 89 esophageal cancer samples and 49 matched adjacent normal esophageal tissues. Results: Six HPV genotypes (HPV6, HPV16, HPV33, HPV39, HPV51, and HPV82) were present in at least 51.7% of the esophageal carcinoma tissues, which was significantly greater than 28.6% prevalence among controls (P < 0.05). HPV16 was the most common of all the genotypes investigated (HPV16 prevalence in carcinoma tissue: 49.4%; odds ratio 3.02, 95% confidence interval 1.39-6.53). HPV-positive ESCC patients were generally younger than HPV-negative patients (P = 0.04). In addition, HPV infection was more common in cases of well-differentiated and shallower invasive depth. Conclusions: Based on this new detection method, our findings reiterate the possibility that HPV infection (especially HPV16) may be involved in the etiology of esophageal carcinoma in the Kazakh populations and that HPV E6 gene positivity may be associated with prognosis of patients. PMID:26722514

  20. The E1B 19,000-molecular-weight protein of group C adenoviruses prevents tumor necrosis factor cytolysis of human cells but not of mouse cells.

    PubMed Central

    Gooding, L R; Aquino, L; Duerksen-Hughes, P J; Day, D; Horton, T M; Yei, S P; Wold, W S

    1991-01-01

    Tumor necrosis factor (TNF) is a multifunctional immunoregulatory protein that is secreted by activated macrophages and is believed to have antiviral activities. We reported earlier that when mouse C3HA fibroblasts are infected with human adenoviruses, the 289R and 243R proteins encoded by region E1A render the cells susceptible to lysis by TNF, and a 14,700-molecular-weight protein (14.7K protein) encoded by region E3 protects the cells against lysis by TNF. We now report that the 19,000-molecular-weight (19K) (176R) protein encoded by the E1B transcription unit can protect human HEL-299 fibroblasts and human ME-180 cervical carcinoma cells against lysis by TNF. This was determined by infecting cells with adenovirus double mutants that lack region E3 and do or do not express the E1B-19K protein and by measuring cytolysis by using a short-term (18-h) 51Cr-release assay. Under these assay conditions, the 51Cr release was specific to TNF and was not a consequence of the cyt phenotype associated with E1B-19K protein-negative mutants. Also, by using virus double mutants that lack E3 in combination with other early regions, we found that E1A, the E1B-55K protein-encoding gene, E3, and E4 are not required to protect HEL-299 cells against TNF cytolysis. Three additional human cancer cell lines (HeLa, HCT8, and RC29) and a simian virus 40-transformed WI38 cell line (VA-13) also required E1B for protection against TNF cytolysis, indicating that the E1B-19K protein is required to protect many if not all human cell types against lysis by TNF when infected by adenovirus. The E1B-19K protein was not able to protect six different adenovirus-infected mouse cell lines against TNF lysis, even though the protein was shown to be efficiently expressed in one of the cell lines. HEL-299 or ME-180 cells infected by a mutant that lacks the E1B-19K protein but retains region E3 were not lysed by TNF, indicating that one or more of the E3 proteins can protect these cells against TNF lysis

  1. Human Papillomavirus Virus (HPV) Genotype- and Age-Specific Analyses of External Genital Lesions Among Men in the HPV Infection in Men (HIM) Study

    PubMed Central

    Ingles, Donna J.; Pierce Campbell, Christine M.; Messina, Jane A.; Stoler, Mark H.; Lin, Hui-Yi; Fulp, William J.; Abrahamsen, Martha; Sirak, Bradley A.; O'Keefe, Michael T.; Papenfuss, Mary; Gage, Christine; Carvalho da Silva, Roberto; Gonzalez Sosa, Rossana; Rojas Juarez, Oscar; Villa, Luisa L.; Lazcano Ponce, Eduardo; Giuliano, Anna R.

    2015-01-01

    Background. Human papillomavirus (HPV) causes external genital lesions (EGLs) in men, including condyloma and penile intraepithelial neoplasia (PeIN). We sought to determine the incidence of pathologically confirmed EGLs, by lesion type, among men in different age groups and to evaluate the HPV types that were associated with EGL development. Methods. HPV Infection in Men (HIM) study participants who contributed ≥2 visits from 2009–2013 were included in the biopsy cohort. Genotyping by an HPV line-probe assay was performed on all pathologically confirmed EGLs. Age-specific analyses were conducted for incident EGLs, with Kaplan–Meier estimation of cumulative incidence. Results. This biopsy cohort included 2754 men (median follow-up duration, 12.4 months [interquartile range, 6.9–19.2 months]). EGLs (n = 377) were pathologically confirmed in 228 men, 198 of whom had incident EGLs. The cumulative incidence of any EGL was highest among men <45 years old and, for condyloma, decreased significantly over time with age. The genotype-specific incidence of EGL varied by pathological diagnoses, with high- and low-risk genotypes found in 15.6% and 73.2% of EGLs, respectively. Condyloma primarily contained HPV 6 or 11. While PeIN lesions primarily contained HPV 16, 1 PeIN III lesion was positive for HPV 6 only. Conclusion. Low- and high-risk HPV genotypes contribute to the EGL burden. Men remain susceptible to HPV-related EGLs throughout the life span, making it necessary to ensure the longevity of immune protection against the most common causative HPV genotypes. PMID:25344518

  2. Polymerase chain reaction (PCR) and sequence specific oligonucleotide probes (SSOP) genotyping assay for detection of genes associated with rheumatoid arthritis and multiple sclerosis.

    PubMed

    Nikolaou, Konstantina; Kalatzis, Fanis G; Giannakeas, Nikolaos; Markoula, Sofia; Chatzikyriakidou, Anthi; Georgiou, Ioannis; Fotiadis, Dimitrios I

    2010-01-01

    In this paper an assay for the detection of genes associated with rheumatoid arthritis (RA) and multiple sclerosis, using polymerase chain reaction (PCR) and sequence specific oligonucleotide probes (SSOP) is presented, in order to be further applied in a portable Lab-On-Chip (LOC) device. A substantial part of these reagents were based on the literature (11th International Histocompatibility Workshop, IHW), bearing the advantage of proven successful implementation in genotyping, while others were designed for this study. More precisely, our methodology discriminates HLA-DRB1 as DRB1*01, *04 and *10, which include shared epitope (SE) alleles associated with RA and additionally DRB1*15 allele, including DRB1*1501 associated with MS (broad genotyping method). To further present the basic elements of the assay for high resolution genotyping of SE DRB1 alleles, we provide as an example the case of HLA-DRB1*10 alleles (HLADRB1* 100101, *100102, *100103, *1002 and *1003). Regarding the methodology for developing a detection assay, for SNPs associated with RA or MS the basic steps are presented. DNA sequence data are obtained from IMGT/HLA and SNP database. Online software tools are used to define hybridization specificity of primers and probes towards human DNA, leading to hybridization patterns that uniquely designate a target allele and evaluate parameters influencing PCR efficiency. Respecting current technological limitations of autonomous molecular-based LOC systems the approach of broad genotyping of HLA-DRB1*01/*04/*10/*15 genes, is intended to be initially used, leaving, high resolution genotyping of SE alleles for future implementations. This method is easy to be updated and extended to detect additional associated loci with RA or MS.

  3. Fruit metabolite networks in engineered and non-engineered tomato genotypes reveal fluidity in a hormone and agroecosystem specific manner

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Multiple strategies have been explored throughout the world to meet food security. These include molecular breeding, transgenic genotype development, reduced-tillage crop production, modification of the soil environment with cover crops or polyethylene mulches and tunnels, and organic farming. Unde...

  4. Novel B19-like parvovirus in the brain of a harbor seal.

    PubMed

    Bodewes, Rogier; Rubio García, Ana; Wiersma, Lidewij C M; Getu, Sarah; Beukers, Martijn; Schapendonk, Claudia M E; van Run, Peter R W A; van de Bildt, Marco W G; Poen, Marjolein J; Osinga, Nynke; Sánchez Contreras, Guillermo J; Kuiken, Thijs; Smits, Saskia L; Osterhaus, Albert D M E

    2013-01-01

    Using random PCR in combination with next-generation sequencing, a novel parvovirus was detected in the brain of a young harbor seal (Phoca vitulina) with chronic non-suppurative meningo-encephalitis that was rehabilitated at the Seal Rehabilitation and Research Centre (SRRC) in the Netherlands. In addition, two novel viruses belonging to the family Anelloviridae were detected in the lungs of this animal. Phylogenetic analysis of the coding sequence of the novel parvovirus, tentatively called Seal parvovirus, indicated that this virus belonged to the genus Erythrovirus, to which human parvovirus B19 also belongs. Although no other seals with similar signs were rehabilitated in SRRC in recent years, a prevalence study of tissues of seals from the same area collected in the period 2008-2012 indicated that the Seal parvovirus has circulated in the harbor seal population at least since 2008. The presence of the Seal parvovirus in the brain was confirmed by real-time PCR and in vitro replication. Using in situ hybridization, we showed for the first time that a parvovirus of the genus Erythrovirus was present in the Virchow-Robin space and in cerebral parenchyma adjacent to the meninges. These findings showed that a parvovirus of the genus Erythrovirus can be involved in central nervous system infection and inflammation, as has also been suspected but not proven for human parvovirus B19 infection.

  5. Novel B19-Like Parvovirus in the Brain of a Harbor Seal

    PubMed Central

    Bodewes, Rogier; Rubio García, Ana; Wiersma, Lidewij C. M.; Getu, Sarah; Beukers, Martijn; Schapendonk, Claudia M. E.; van Run, Peter R. W. A.; van de Bildt, Marco W. G.; Poen, Marjolein J.; Osinga, Nynke; Sánchez Contreras, Guillermo J.; Kuiken, Thijs; Smits, Saskia L.; Osterhaus, Albert D. M. E.

    2013-01-01

    Using random PCR in combination with next-generation sequencing, a novel parvovirus was detected in the brain of a young harbor seal (Phoca vitulina) with chronic non-suppurative meningo-encephalitis that was rehabilitated at the Seal Rehabilitation and Research Centre (SRRC) in the Netherlands. In addition, two novel viruses belonging to the family Anelloviridae were detected in the lungs of this animal. Phylogenetic analysis of the coding sequence of the novel parvovirus, tentatively called Seal parvovirus, indicated that this virus belonged to the genus Erythrovirus, to which human parvovirus B19 also belongs. Although no other seals with similar signs were rehabilitated in SRRC in recent years, a prevalence study of tissues of seals from the same area collected in the period 2008-2012 indicated that the Seal parvovirus has circulated in the harbor seal population at least since 2008. The presence of the Seal parvovirus in the brain was confirmed by real-time PCR and in vitro replication. Using in situ hybridization, we showed for the first time that a parvovirus of the genus Erythrovirus was present in the Virchow-Robin space and in cerebral parenchyma adjacent to the meninges. These findings showed that a parvovirus of the genus Erythrovirus can be involved in central nervous system infection and inflammation, as has also been suspected but not proven for human parvovirus B19 infection. PMID:24223918

  6. Improved loop-mediated isothermal amplification for HLA-DRB1 genotyping using RecA and a restriction enzyme for enhanced amplification specificity.

    PubMed

    Mitsunaga, Shigeki; Shimizu, Sayoko; Okudaira, Yuko; Oka, Akira; Tanaka, Masafumi; Kimura, Minoru; Kulski, Jerzy K; Inoue, Ituro; Inoko, Hidetoshi

    2013-06-01

    Our aim was to test and develop the use of loop-mediated isothermal amplification (LAMP) for HLA-DRB1 genotyping. Initially, we found that the conventional LAMP protocols produced non-specific and variable amplification results depending on the sample DNA conditions. Experiments with different concentrations of DNase in the reaction mixture with and without T4 DNA ligase-treated samples suggested that the strand displacement activity of DNA polymerase in LAMP, at least in part, started from randomly existing nicks because T4 DNA ligase treatment of sample DNA resulted in no amplification. Such non-specific amplification due to the randomly existing nicks was improved specifically by the addition of RecA of Escherichia coli and a restriction enzyme, for example, PvuII, to the reaction mixture. We applied the modified LAMP (mLAMP) (1) to detect specific HLA-DRB1 alleles by using only specific primers for amplification or (2) for genotyping in multiple samples with a multi-probe typing system. In the latter case, HLA-DRB1 genotyping was developed by combining the mLAMP with amplicon capture using polymorphic region-specific probes fixed onto the bottom of the wells of a 96-well plate and the captured amplicons visualized as a black spot at the bottom of the well. The multi-probe human leukocyte antigen (HLA) typing method and the specific HLA allele detection method could be applied for point-of-care testing due to no requirement for specific and expensive instruments.

  7. Identification of a novel simian parvovirus in cynomolgus monkeys with severe anemia. A paradigm of human B19 parvovirus infection.

    PubMed Central

    O'Sullivan, M G; Anderson, D C; Fikes, J D; Bain, F T; Carlson, C S; Green, S W; Young, N S; Brown, K E

    1994-01-01

    Although human B19 parvovirus infection has been clearly associated with a number of distinct syndromes (including severe anemia, abortion, and arthritis), detailed knowledge of its pathogenesis has been hindered by the lack of a suitable animal model. We have identified a novel simian parvovirus in cynomolgus monkeys with severe anemia. Sequencing of a 723-bp fragment of cloned viral DNA extracted from serum revealed that the simian parvovirus has 65% homology at the DNA level with the human B19 parvovirus but little homology with other known parvoviruses. Light microscopic examination of bone marrow from infected animals showed intranuclear inclusion bodies, and ultrastructural studies showed viral arrays characteristic of parvoviruses. Another striking feature was the presence of marked dyserythropoiesis in cells of the erythroid lineage, raising the possibility that B19 parvovirus infection may underlie related dyserythropoietic syndromes in human beings. Affected animals had concurrent infection with the immunosuppressive type D simian retrovirus, analogous to HIV patients who develop severe anemia because of infection with B19 parvovirus. The remarkable similarities between the simian and B19 parvoviruses suggest that experimentally infected cynomolgus monkeys may serve as a useful animal model of human B19 infection. Images PMID:8163659

  8. Differential response of tomato genotypes to Xanthomonas-specific pathogen-associated molecular patterns and correlation with bacterial spot (Xanthomonas perforans) resistance

    PubMed Central

    Bhattarai, Krishna; Louws, Frank J; Williamson, John D; Panthee, Dilip R

    2016-01-01

    Plants depend on innate immune responses to retard the initial spread of pathogens entering through stomata, hydathodes or injuries. These responses are triggered by conserved patterns in pathogen-encoded molecules known as pathogen-associated molecular patterns (PAMPs). Production of reactive oxygen species (ROS) is one of the first responses, and the resulting ‘oxidative burst’ is considered to be a first line of defense. In this study, we conducted association analyses between ROS production and bacterial spot (BS; Xanthomonas spp.) resistance in 63 genotypes of tomato (Solanum lycopersicum L.). A luminol-based assay was performed on leaf tissues that had been treated with a flagellin 22 (flg22), flagellin 28 and a Xanthomonas-specific flg22 (flg22-Xac) peptide, to measure PAMP-induced ROS production in each genotype. These genotypes were also assessed for BS disease response by inoculation with Xanthomonas perforans, race T4. Although there was no consistent relationship between peptides used and host response to the BS, there was a significant negative correlation (r=−0.25, P<0.05) between foliar disease severity and ROS production, when flg22-Xac was used. This response could potentially be used to identify the Xanthomonas-specific PRR allele in tomato, and eventually PAMP-triggered immunity loci could be mapped in a segregating population. This has potential significance in tomato improvement. PMID:27555919

  9. Differential response of tomato genotypes to Xanthomonas-specific pathogen-associated molecular patterns and correlation with bacterial spot (Xanthomonas perforans) resistance.

    PubMed

    Bhattarai, Krishna; Louws, Frank J; Williamson, John D; Panthee, Dilip R

    2016-01-01

    Plants depend on innate immune responses to retard the initial spread of pathogens entering through stomata, hydathodes or injuries. These responses are triggered by conserved patterns in pathogen-encoded molecules known as pathogen-associated molecular patterns (PAMPs). Production of reactive oxygen species (ROS) is one of the first responses, and the resulting 'oxidative burst' is considered to be a first line of defense. In this study, we conducted association analyses between ROS production and bacterial spot (BS; Xanthomonas spp.) resistance in 63 genotypes of tomato (Solanum lycopersicum L.). A luminol-based assay was performed on leaf tissues that had been treated with a flagellin 22 (flg22), flagellin 28 and a Xanthomonas-specific flg22 (flg22-Xac) peptide, to measure PAMP-induced ROS production in each genotype. These genotypes were also assessed for BS disease response by inoculation with Xanthomonas perforans, race T4. Although there was no consistent relationship between peptides used and host response to the BS, there was a significant negative correlation (r=-0.25, P<0.05) between foliar disease severity and ROS production, when flg22-Xac was used. This response could potentially be used to identify the Xanthomonas-specific PRR allele in tomato, and eventually PAMP-triggered immunity loci could be mapped in a segregating population. This has potential significance in tomato improvement. PMID:27555919

  10. Production of viable male unreduced gametes in Brassica interspecific hybrids is genotype specific and stimulated by cold temperatures

    PubMed Central

    2011-01-01

    Background Unreduced gametes (gametes with the somatic chromosome number) may provide a pathway for evolutionary speciation via allopolyploid formation. We evaluated the effect of genotype and temperature on male unreduced gamete formation in Brassica allotetraploids and their interspecific hybrids. The frequency of unreduced gametes post-meiosis was estimated in sporads from the frequency of dyads or giant tetrads, and in pollen from the frequency of viable giant pollen compared with viable normal pollen. Giant tetrads were twice the volume of normal tetrads, and presumably resulted from pre-meiotic doubling of chromosome number. Giant pollen was defined as pollen with more than 1.5 × normal diameter, under the assumption that the doubling of DNA content in unreduced gametes would approximately double the pollen cell volume. The effect of genotype was assessed in five B. napus, two B. carinata and one B. juncea parents and in 13 interspecific hybrid combinations. The effect of temperature was assessed in a subset of genotypes in hot (day/night 30°C/20°C), warm (25°C/15°C), cool (18°C/13°C) and cold (10°C/5°C) treatments. Results Based on estimates at the sporad stage, some interspecific hybrid genotypes produced unreduced gametes (range 0.06 to 3.29%) at more than an order of magnitude higher frequency than in the parents (range 0.00% to 0.11%). In nine hybrids that produced viable mature pollen, the frequency of viable giant pollen (range 0.2% to 33.5%) was much greater than in the parents (range 0.0% to 0.4%). Giant pollen, most likely formed from unreduced gametes, was more viable than normal pollen in hybrids. Two B. napus × B. carinata hybrids produced 9% and 23% unreduced gametes based on post-meiotic sporad observations in the cold temperature treatment, which was more than two orders of magnitude higher than in the parents. Conclusions These results demonstrate that sources of unreduced gametes, required for the triploid bridge hypothesis of

  11. Male-specific genotype by environment interactions influence viability selection acting on a sexually selected inversion system in the seaweed fly, Coelopa frigida.

    PubMed

    Edward, Dominic A; Gilburn, André S

    2013-01-01

    In the seaweed fly, Coelopa frigida, a large chromosomal inversion system is affected by sexual selection and viability selection. However, our understanding of the interaction between these two selective forces is currently limited as research has focused upon a limited range of environments. We allowed C. frigida larvae to develop in two different algae, Fucus and Laminaria, and then measured viability and body size for each inversion genotype. Significant male-specific genotype-by-environment interactions influenced viability and body size. For males developing in Laminaria, the direction of viability selection acts similarly on the inversion system as the direction of sexual selection. In contrast, for males developing in Fucus, viability selection opposes sexual selection. These results demonstrate that through considering viability selection in different environments, the costs and benefits associated with sexual selection can be found to vary.

  12. Photosystem II thermostability in situ: environmentally induced acclimation and genotype-specific reactions in Triticum aestivum L.

    PubMed

    Brestic, Marian; Zivcak, Marek; Kalaji, Hazem M; Carpentier, Robert; Allakhverdiev, Suleyman I

    2012-08-01

    Photosystem II (PSII) thermostability and acclimation effects on PSII photochemical efficiency were analyzed in thirty field grown winter wheat (Triticum aestivum L.) genotypes using prompt chlorophyll a fluorescence kinetics before and after dark heat treatment. A gradual increase in temperature caused the appearance of K-bands at 300 μs on the chlorophyll fluorescence induction curve, indicating the impairment of the PSII donor side (even by heat treatment at 38 °C). An increase in basal fluorescence, commonly used as a criterion of PSII thermostability, was observed beyond a temperature threshold of 44 °C. Moreover, an acclimation shift (increase of critical temperature) was observed at the 3.5 °C identified for K-band appearance, but only by 1.1 °C for a steep increase in F(0). The single temperature approach with regular weekly observations completed within two months using dark heat treatment at 40 °C demonstrated that the acclimation effect is not gradual, but occurs immediately and is associated with an increase of daily temperature maxima over 30 °C. The acclimated heat treated samples had less effect on the donor side of PSII, the higher fraction of active Q(A)(-) reducing reaction centers and causing a much lower decrease of connectivity among PSII units compared to non-acclimated samples. In the non-treated plants the reduction of antennae size, increase of PSII connectivity and changes in the acceptor side occurred as a result of heat acclimation. The enhancement of PSII thermostability persisted over several weeks regardless of weather conditions. The genotype comparison identified three groups that differed either in initial PSII thermostability or in acclimation capacity; these groupings were clearly associated with the origin of the genotypes.

  13. Disagreement in genotyping results of drug resistance alleles of the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) gene by allele-specific PCR (ASPCR) assays and Sanger sequencing.

    PubMed

    Sharma, Divya; Lather, Manila; Dykes, Cherry L; Dang, Amita S; Adak, Tridibes; Singh, Om P

    2016-01-01

    The rapid spread of antimalarial drug resistance in Plasmodium falciparum over the past few decades has necessitated intensive monitoring of such resistance for an effective malaria control strategy. P. falciparum dihydropteroate synthase (Pfdhps) and P. falciparum dihydrofolate reductase (Pfdhfr) genes act as molecular markers for resistance against the antimalarial drugs sulphadoxine and pyrimethamine, respectively. Resistance to pyrimethamine which is used as a partner drug in artemisinin combination therapy (ACT) is associated with several mutations in the Pfdhfr gene, namely A16V, N51I, C59R, S108N/T and I164L. Therefore, routine monitoring of Pfdhfr-drug-resistant alleles in a population may help in effective drug resistance management. Allele-specific PCR (ASPCR) is one of the commonly used methods for molecular genotyping of these alleles. In this study, we genotyped 55 samples of P. falciparum for allele discrimination at four codons of Pfdhfr (N51, C59, S108 and I164) by ASPCR using published methods and by Sanger's DNA sequencing method. We found that the ASPCR identified a significantly higher number of mutant alleles as compared to the DNA sequencing method. Such discrepancies arise due to the non-specificity of some of the allele-specific primer sets and due to the lack of sensitivity of Sanger's DNA sequencing method to detect minor alleles present in multiple clone infections. This study reveals the need of a highly specific and sensitive method for genotyping and detecting minor drug-resistant alleles present in multiple clonal infections.

  14. Disagreement in genotyping results of drug resistance alleles of the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) gene by allele-specific PCR (ASPCR) assays and Sanger sequencing.

    PubMed

    Sharma, Divya; Lather, Manila; Dykes, Cherry L; Dang, Amita S; Adak, Tridibes; Singh, Om P

    2016-01-01

    The rapid spread of antimalarial drug resistance in Plasmodium falciparum over the past few decades has necessitated intensive monitoring of such resistance for an effective malaria control strategy. P. falciparum dihydropteroate synthase (Pfdhps) and P. falciparum dihydrofolate reductase (Pfdhfr) genes act as molecular markers for resistance against the antimalarial drugs sulphadoxine and pyrimethamine, respectively. Resistance to pyrimethamine which is used as a partner drug in artemisinin combination therapy (ACT) is associated with several mutations in the Pfdhfr gene, namely A16V, N51I, C59R, S108N/T and I164L. Therefore, routine monitoring of Pfdhfr-drug-resistant alleles in a population may help in effective drug resistance management. Allele-specific PCR (ASPCR) is one of the commonly used methods for molecular genotyping of these alleles. In this study, we genotyped 55 samples of P. falciparum for allele discrimination at four codons of Pfdhfr (N51, C59, S108 and I164) by ASPCR using published methods and by Sanger's DNA sequencing method. We found that the ASPCR identified a significantly higher number of mutant alleles as compared to the DNA sequencing method. Such discrepancies arise due to the non-specificity of some of the allele-specific primer sets and due to the lack of sensitivity of Sanger's DNA sequencing method to detect minor alleles present in multiple clone infections. This study reveals the need of a highly specific and sensitive method for genotyping and detecting minor drug-resistant alleles present in multiple clonal infections. PMID:26407876

  15. AN EVALUATION OF CRYPTOSPORIDIUM PARVUM GENOTYPING

    EPA Science Inventory

    We evaluated the specificity and sensitivity of 11 previously described species differentiation and genotyping PCR protocols for detection of Cryptosporidium parasites. Genomic DNA from three species of Crytosporidium parasites (genotype 1 and genotype 2 of C. parvum, C. muris, a...

  16. Nucleotide sequence and genome organization of human parvovirus B19 isolated from the serum of a child during aplastic crisis.

    PubMed

    Shade, R O; Blundell, M C; Cotmore, S F; Tattersall, P; Astell, C R

    1986-06-01

    The nucleotide sequence of an almost-full-length clone of human parvovirus B19 was determined. Whereas the extreme left and right ends of this genomic clone are incomplete, the sequence clearly indicates that the two ends of viral DNA are related by inverted terminal repeats similar to those of the Dependovirus genus. The coding regions are complete in the cloned DNA, and the two large open reading frames which span almost the entire genome are restricted to one strand, as has been found for all other parvoviruses characterized to date. From the DNA sequence we conclude that the organization of the B19 transcription units is similar although not identical to those of other parvoviruses. In particular, we predict that the B19 genome may utilize a fourth promoter to transcribe mRNA encoding the major structural polypeptide, VP2. Analysis of the putative polypeptides confirms that B19 is only distantly related to the other parvoviruses but reveals that there is a small region in the gene probably encoding the major nonstructural protein of B19, which is closely conserved between all of the parvovirus genomes for which sequence information is currently available.

  17. Study of Mycobacterium tuberculosis Complex Genotypic Diversity in Malaysia Reveals a Predominance of Ancestral East-African-Indian Lineage with a Malaysia-Specific Signature

    PubMed Central

    Ismail, Fazli; Couvin, David; Farakhin, Izzah; Abdul Rahman, Zaidah; Rastogi, Nalin; Suraiya, Siti

    2014-01-01

    Background Tuberculosis (TB) still constitutes a major public health problem in Malaysia. The identification and genotyping based characterization of Mycobacterium tuberculosis complex (MTBC) isolates causing the disease is important to determine the effectiveness of the control and surveillance programs. Objectives This study intended a first assessment of spoligotyping-based MTBC genotypic diversity in Malaysia followed by a comparison of strains with those prevailing in neighboring countries by comparison with an international MTBC genotyping database. Methods Spoligotyping was performed on a total of 220 M. tuberculosis clinical isolates collected in Kelantan and Kuala Lumpur. The results were compared with the SITVIT2 international database of the Pasteur Institute of Guadeloupe. Results Spoligotyping revealed 77 different patterns: 22 corresponded to orphan patterns while 55 patterns containing 198 isolates were assigned a Spoligo International Type (SIT) designation in the database (the latter included 6 newly created SITs). The eight most common SITs grouped 141 isolates (5 to 56 strains per cluster) as follows: SIT1/Beijing, n = 56, 25.5%; SIT745/EAI1-SOM, n = 33, 15.0%; SIT591/EAI6-BGD1, n = 13, 5.9%; SIT256/EAI5, n = 12, 5.5%; SIT236/EAI5, n = 10, 4.6%; SIT19/EAI2-Manila, n = 9, 4.1%; SIT89/EAI2-Nonthaburi, n = 5, 2.3%; and SIT50/H3, n = 3, 1.4%. The association between city of isolation and lineages was statistically significant; Haarlem and T lineages being higher in Kuala Lumpur (p<0.01). However, no statistically significant differences were noted when comparing drug resistance vs. major lineages, nor between gender and clades. Conclusions The ancestral East-African-Indian (EAI) lineage was most predominant followed by the Beijing lineage. A comparison of strains with those prevailing in neighboring countries in South Asia, East Asia and South East Asia underlined the phylogeographical specificity of SIT745 for

  18. The effects of co-infection with human parvovirus B19 and Plasmodium falciparum on type and degree of anaemia in Ghanaian children

    PubMed Central

    Duedu, Kwabena Obeng; Sagoe, Kwamena William Coleman; Ayeh-Kumi, Patrick Ferdinand; Affrim, Raymond Bedu; Adiku, Theophilus

    2013-01-01

    Objective To determin the extent to which parvovirus B19 (B19V) and co-infection of B19V and malaria contribute to risk of anaemia in children. Methods B19V DNA and malaria parasites were screened for 234 children at the PML Children's Hospital in Accra. The role of B19V and co-infection with B19V and malaria in anaemia was evaluated by analysing full blood cell counts, malaria and B19V DNA results from these children. Results The prevalence of B19V, malaria and co-infection with B19V and malaria was 4.7%, 41.9% and 2.6%, respectively. Malaria posed a greater risk in the development of mild anaemia compared to severe anaemia (OR=5.28 vrs 3.15) whereas B19V posed a higher risk in the development of severe anaemia compared to mild anaemia (OR=4.07 vrs 1.00) from a non-anaemic child. Persons with co-infection with B19V and malaria had 2.23 times the risk (95% CI=0.40-12.54) of developing severe anaemia should they already have a mild anaemia. The degree of anaemia was about three times affected by co-infection (Pillai's trace=0.551, P=0.001) as was affected by malaria alone (Pillai's trace=0.185, P=0.001). B19V alone did not significantly affect the development of anaemia in a non-anaemic child. Microcytic anaemia was associated with B19V and co-infection with B19V and malaria more than normocytic normochromic anaemia. Conclusions B19V was associated with malaria in cases of severe anaemia. The association posed a significant risk for exacerbation of anaemia in mild anaemic children. B19V and co-infection with B19V and malaria may be associated with microcytic anaemia rather than normocytic normochromic anaemia as seen in cases of B19V infection among persons with red cell abnormalities. PMID:23593592

  19. Sex-specific genotype-by-environment interactions for cuticular hydrocarbon expression in decorated crickets, Gryllodes sigillatus: implications for the evolution of signal reliability.

    PubMed

    Weddle, C B; Mitchell, C; Bay, S K; Sakaluk, S K; Hunt, J

    2012-10-01

    Phenotypic traits that convey information about individual identity or quality are important in animal social interactions, and the degree to which such traits are influenced by environmental variation can have profound effects on the reliability of these cues. Using inbred genetic lines of the decorated cricket, Gryllodes sigillatus, we manipulated diet quality to test how the cuticular hydrocarbon (CHC) profiles of males and females respond across two different nutritional rearing environments. There were significant differences between lines in the CHC profiles of females, but the effect of diet was not quite statistically significant. There was no significant genotype-by-environment interaction (GEI), suggesting that environmental effects on phenotypic variation in female CHCs are independent of genotype. There was, however, a significant effect of GEI for males, with changes in both signal quantity and content, suggesting that environmental effects on phenotypic expression of male CHCs are dependent on genotype. The differential response of male and female CHC expression to variation in the nutritional environment suggests that these chemical cues may be under sex-specific selection for signal reliability. Female CHCs show the characteristics of reliable cues of identity: high genetic variability, low condition dependence and a high degree of genetic determination. This supports earlier work showing that female CHCs are used in self-recognition to identify previous mates and facilitate polyandry. In contrast, male CHCs show the characteristics of reliable cues of quality: condition dependence and a relatively higher degree of environmental determination. This suggests that male CHCs are likely to function as cues of underlying quality during mate choice and/or male dominance interactions.

  20. Coproduction of KPC-2 and QnrB19 in Klebsiella pneumoniae ST340 isolate in Brazil.

    PubMed

    Martins, Willames M B S; Almeida, Anna C S; Nicoletti, Adriana G; Cayô, Rodrigo; Gales, Ana C; Alves, Luiz C; Brayner, Fábio B; Vilela, Marinalda A; Morais, Márcia M C

    2015-12-01

    Few reports described the presence of bla(KPC) and qnr genes in the same isolate. This study reports the combination of bla(KPC-2) and qnrB19 genes in Klebsiella pneumoniae ST340 isolate in Brazil. These findings draw attention to this combination in ST340 isolate, which is part of the CC258, disseminated in Latin America. PMID:26458280

  1. 75 FR 69609 - Airworthiness Directives; Bombardier, Inc. Model CL-600-2B19 (Regional Jet Series 100 & 440...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-15

    ... in production on aircraft Serial Numbers (S/N) 7213 and subsequent. Service Bulletin 601R-30-022... 12866; 2. Is not a ``significant rule'' under the DOT Regulatory Policies and Procedures (44 FR 11034...-600-2B19 (Regional Jet Series 100 & 440) airplanes; certificated in any category; serial numbers...

  2. 75 FR 22521 - Airworthiness Directives; Bombardier, Inc. Model CL-600-2B19 (Regional Jet Series 100 & 440...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-29

    .... Model CL-600-2B19 (Regional Jet Series 100 & 440) Airplanes AGENCY: Federal Aviation Administration (FAA... transportation, Aircraft, Aviation safety, Incorporation by reference, Safety. Adoption of the Amendment 0... Federal Aviation Administration 14 CFR Part 39 [Docket No. FAA-2009-0525; Directorate Identifier...

  3. 75 FR 10667 - Airworthiness Directives; Bombardier, Inc. Model CL-600-2B19 (Regional Jet Series 100 & 440...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-09

    ..., Inc. Model CL-600-2B19 (Regional Jet Series 100 & 440) Airplanes AGENCY: Federal Aviation... the AD docket. List of Subjects in 14 CFR Part 39 Air transportation, Aircraft, Aviation safety... TRANSPORTATION Federal Aviation Administration 14 CFR Part 39 [Docket No. FAA-2010-0178; Directorate...

  4. 75 FR 64960 - Airworthiness Directives; Bombardier, Inc. Model CL-600-2B19 (Regional Jet Series 100 & 440...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-21

    ... Directives; Bombardier, Inc. Model CL-600-2B19 (Regional Jet Series 100 & 440) Airplanes AGENCY: Federal... Regional Jet engine TCGB P/Ns: 2100140-003, 2100140- 005 & 2100140-007. Some of these failures have... too close to the fuel shut-off position, which potentially, can cause the engine to flame...

  5. 76 FR 10216 - Airworthiness Directives; Bombardier, Inc. Model CL-600-2B19 (Regional Jet Series 100 & 440...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-02-24

    .... Model CL-600-2B19 (Regional Jet Series 100 & 440) Airplanes AGENCY: Federal Aviation Administration (FAA... been numerous reported failures of the Regional Jet engine TCGB P/Ns: 2100140-003, 2100140- 005... idle position can result in throttle moving too close to the fuel shut-off position, which...

  6. 75 FR 64636 - Airworthiness Directives; Bombardier, Inc. Model CL-600-2B19 (Regional Jet Series 100 & 440...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-20

    ... provided by paragraph(s) of this AD, no alternative actions specified in Canadair Regional Jet TR RJ/186-1.... Model CL-600-2B19 (Regional Jet Series 100 & 440) Airplanes AGENCY: Federal Aviation Administration (FAA... Jet Temporary Revision (TR) RJ/186-1, dated August 24, 2010, to the Limitations section,...

  7. 75 FR 34657 - Airworthiness Directives; Bombardier, Inc. Model CL-600-2B19 (Regional Jet Series 100 & 440...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-18

    ... and generator housing assembly, have been reported on CL-600-2B19 aircraft. The same part is also installed on CL-600-2C10, -2D15 and -2D24 aircraft. Investigation determined that the crack was in an area... possibly result in damage to the aircraft structure. If deployment was activated by a dual engine...

  8. Coproduction of KPC-2 and QnrB19 in Klebsiella pneumoniae ST340 isolate in Brazil.

    PubMed

    Martins, Willames M B S; Almeida, Anna C S; Nicoletti, Adriana G; Cayô, Rodrigo; Gales, Ana C; Alves, Luiz C; Brayner, Fábio B; Vilela, Marinalda A; Morais, Márcia M C

    2015-12-01

    Few reports described the presence of bla(KPC) and qnr genes in the same isolate. This study reports the combination of bla(KPC-2) and qnrB19 genes in Klebsiella pneumoniae ST340 isolate in Brazil. These findings draw attention to this combination in ST340 isolate, which is part of the CC258, disseminated in Latin America.

  9. 76 FR 38065 - Airworthiness Directives; Bombardier, Inc. Model CL-600-2B19 (Regional Jet Series 100 & 440...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-29

    ... experienced on CL-600-2B19 aeroplanes, resulting in the loss of the associated hydraulic system and high... 2010-22-02 did not require the removal of the hydraulic system No. 3 accumulator, or replacement of the hydraulic system No. 1, inboard brake, and outboard brake accumulators, as specified in Part IV and Part...

  10. De novo transcriptome assembly and analysis of differentially expressed genes of two barley genotypes reveal root-zone-specific responses to salt exposure

    PubMed Central

    Hill, Camilla Beate; Cassin, Andrew; Keeble-Gagnère, Gabriel; Doblin, Monika S.; Bacic, Antony; Roessner, Ute

    2016-01-01

    Plant roots are the first organs sensing and responding to salinity stress, manifested differentially between different root types, and also at the individual tissue and cellular level. High genetic diversity and the current lack of an assembled map-based sequence of the barley genome severely limit barley research potential. We used over 580 and 600 million paired-end reads, respectively, to create two de novo assemblies of a barley landrace (Sahara) and a malting cultivar (Clipper) with known contrasting responses to salinity. Generalized linear models were used to statistically access spatial, treatment-related, and genotype-specific responses. This revealed a spatial gene expression gradient along the barley root, with more differentially expressed transcripts detected between different root zones than between treatments. The root transcriptome also showed a gradual transition from transcripts related to sugar-mediated signaling at the root meristematic zone to those involved in cell wall metabolism in the elongation zone, and defense response-related pathways toward the maturation zone, with significant differences between the two genotypes. The availability of these additional transcriptome reference sets will serve as a valuable resource to the cereal research community, and may identify valuable traits to assist in breeding programmes. PMID:27527578

  11. De novo transcriptome assembly and analysis of differentially expressed genes of two barley genotypes reveal root-zone-specific responses to salt exposure.

    PubMed

    Hill, Camilla Beate; Cassin, Andrew; Keeble-Gagnère, Gabriel; Doblin, Monika S; Bacic, Antony; Roessner, Ute

    2016-01-01

    Plant roots are the first organs sensing and responding to salinity stress, manifested differentially between different root types, and also at the individual tissue and cellular level. High genetic diversity and the current lack of an assembled map-based sequence of the barley genome severely limit barley research potential. We used over 580 and 600 million paired-end reads, respectively, to create two de novo assemblies of a barley landrace (Sahara) and a malting cultivar (Clipper) with known contrasting responses to salinity. Generalized linear models were used to statistically access spatial, treatment-related, and genotype-specific responses. This revealed a spatial gene expression gradient along the barley root, with more differentially expressed transcripts detected between different root zones than between treatments. The root transcriptome also showed a gradual transition from transcripts related to sugar-mediated signaling at the root meristematic zone to those involved in cell wall metabolism in the elongation zone, and defense response-related pathways toward the maturation zone, with significant differences between the two genotypes. The availability of these additional transcriptome reference sets will serve as a valuable resource to the cereal research community, and may identify valuable traits to assist in breeding programmes. PMID:27527578

  12. A Sorghum bicolor expression atlas reveals dynamic genotype-specific expression profiles for vegetative tissues of grain, sweet and bioenergy sorghums

    SciTech Connect

    Shakoor, N; Nair, R; Crasta, O; Morris, G; Feltus, A; Kresovich, S

    2014-01-23

    Background: Effective improvement in sorghum crop development necessitates a genomics-based approach to identify functional genes and QTLs. Sequenced in 2009, a comprehensive annotation of the sorghum genome and the development of functional genomics resources is key to enable the discovery and deployment of regulatory and metabolic genes and gene networks for crop improvement. Results: This study utilizes the first commercially available whole-transcriptome sorghum microarray (Sorgh-WTa520972F) to identify tissue and genotype-specific expression patterns for all identified Sorghum bicolor exons and UTRs. The genechip contains 1,026,373 probes covering 149,182 exons (27,577 genes) across the Sorghum bicolor nuclear, chloroplast, and mitochondrial genomes. Specific probesets were also included for putative non-coding RNAs that may play a role in gene regulation (e. g., microRNAs), and confirmed functional small RNAs in related species (maize and sugarcane) were also included in our array design. We generated expression data for 78 samples with a combination of four different tissue types (shoot, root, leaf and stem), two dissected stem tissues (pith and rind) and six diverse genotypes, which included 6 public sorghum lines (R159, Atlas, Fremont, PI152611, AR2400 and PI455230) representing grain, sweet, forage, and high biomass ideotypes. Conclusions: Here we present a summary of the microarray dataset, including analysis of tissue-specific gene expression profiles and associated expression profiles of relevant metabolic pathways. With an aim to enable identification and functional characterization of genes in sorghum, this expression atlas presents a new and valuable resource to the research community.

  13. Quantitative analysis of human parvovirus B19 DNA in maternal and fetal serum, and amniotic fluid during an early stage of pregnancy.

    PubMed

    Ishikawa, Aki; Yoto, Yuko; Asakura, Hirofumi; Tsutsumi, Hiroyuki

    2015-04-01

    Simple and accurate diagnosis of vertical transmission of human parvovirus B19 (B19V) infection remains an important issue in pregnancy. There are few reports on quantitative analysis of B19V in amniotic fluids. Quantitative estimation of B19V DNA in amniotic fluids was comparerd with those in maternal or fetal serum obtained at an early stage of pregnancy with possible mother-to-fetus transmission. All pregnant women contracted B19V infection between 13 to 14 weeks gestation. The B19V DNA amount in 3 maternal serum and amniotic fluid sample pairs collected between 16 to 27 weeks gestation was quantified by a real-time polymerase chain reaction assay. Serum from 2 fetuses was included. The B19V DNA concentrations in maternal sera and amniotic fluids ranged from 10(4) to 10(5) copies/ml and from 10(7) to 10(8) copies/ml, respectively. The B19V DNA in the amniotic fluids concentration coincided with those of each fetal serum. The concentrations in amniotic fluids are 100 to 5,000 times higher than in those of maternal sera, and corresponded to the matching fetal serum. Amniotic fluids may substitute for the fetal sera in terms of quantitative estimation of fetal B19V infection at an early stage of pregnancy.

  14. Increased expression of Matrix Metalloproteinase 9 in liver from NZB/W F1 mice received antibody against human parvovirus B19 VP1 unique region protein

    PubMed Central

    Tsai, Chun-Chou; Tzang, Bor-Show; Chiang, Szu-Yi; Hsu, Gwo-Jong; Hsu, Tsai-Ching

    2009-01-01

    Background Human parvovirus B19 infection has been postulated to the anti-phospholipid syndrome (APS) in autoimmunity. However, the influence of anti-B19-VP1u antibody in autoimmune diseases is still obscure. Methods To elucidate the effect of anti-B19-VP1u antibodies in systemic lupus erythematosus (SLE), passive transfer of rabbit anti-B19-VP1u IgG was injected intravenously into NZB/W F1 mice. Results Significant reduction of platelet count and prolonged thrombocytopenia time were detected in anti-B19-VP1u IgG group as compared to other groups, whereas significant increases of anti-B19-VP1u, anti-phospholipid (APhL), and anti-double strand DNA (dsDNA) antibody binding activity were detected in anti-B19-VP1u group. Additionally, significant increases of matrix metalloproteinase-9 (MMP9) activity and protein expression were detected in B19-VP1u IgG group. Notably, phosphatidylinositol 3-phosphate kinase (PI3K) and phosphorylated extracellular signal-regulated kinase (ERK) proteins were involved in the induction of MMP9. Conclusion These experimental results firstly demonstrated the aggravated effects of anti-B19-VP1u antibody in disease activity of SLE. PMID:19272186

  15. Effects of B2/B19‧ phase boundary on thermally induced phase transition in NiTi: An atomistic study

    NASA Astrophysics Data System (ADS)

    Qin, Sheng-Jian; Shang, Jia-Xiang; Wang, Xu; Wang, Fu-He

    2015-10-01

    Molecular dynamics simulations are performed to study the thermally induced phase transition in NiTi B19‧ single crystal at high temperatures (400, 500, 600 and 700 K), B2 single crystal at low temperatures (50, 100, 150 and 200 K) and the system containing a B2/B19‧ phase boundary at high, medium (300, 320 and 340 K) and low temperatures. Homogeneous nucleation and growth of the B2 and the B19‧ phases are observed in B19‧ and B2 single crystal systems, respectively. In B2/B19‧ system, heterogeneous nucleation and growth of the B2 phase are observed at high temperatures, which results in forming a B2/B2 grain boundary and a new B2/B19‧ phase boundary; the B2 phase transforms homogeneously to B19‧ phase at low temperatures; the B2 and B19‧ phases coexist without heterogeneous nucleation of any phase at medium temperatures. Compared with the single crystal, the B2/B19‧ phase boundary provides the sites for heterogeneous B2 nucleation at high temperatures and has little effect on the phase transition at low temperatures.

  16. Children's 5-HTTLPR genotype moderates the link between maternal criticism and attentional biases specifically for facial displays of anger.

    PubMed

    Gibb, Brandon E; Johnson, Ashley L; Benas, Jessica S; Uhrlass, Dorothy J; Knopik, Valerie S; McGeary, John E

    2011-09-01

    Theorists have proposed that negative experiences in childhood may contribute to the development of experience-specific information-processing biases, including attentional biases. There are also clear genetic influences on cognitive processes, with evidence that polymorphisms in specific candidate genes may moderate the impact of environmental stress on attentional biases (e.g., a functional polymorphism in the serotonin transporter gene; 5-HTTLPR). In the current study, we tested a gene×environment (G×E) model of risk for attentional biases. We hypothesised that children whose mothers exhibit high levels of expressed emotion criticism (EE-Crit) would display attentional biases specifically for angry, but not happy or sad, faces, and that this link would be stronger among children carrying one or two copies of the 5-HTTLPR short allele than among those homozygous for the long allele. Results generally supported these hypotheses, though we found that carriers of the 5-HTTLPR short allele who also had a critical mother exhibited attentional avoidance of angry faces rather than preferential attention.

  17. Experimental evolution of parasitoid infectivity on symbiont-protected hosts leads to the emergence of genotype specificity.

    PubMed

    Rouchet, Romain; Vorburger, Christoph

    2014-06-01

    Host-parasitoid interactions may lead to strong reciprocal selection for traits involved in host defense and parasitoid counterdefense. In aphids, individuals harboring the facultative bacterial endosymbiont, Hamiltonella defensa, exhibit enhanced resistance to parasitoid wasps. We used an experimental evolution approach to investigate the ability of the parasitoid wasp, Lysiphlebus fabarum, to adapt to the presence of H. defensa in its aphid host Aphis fabae. Sexual populations of the parasitoid were exposed for 11 generations to a single clone of A. fabae, either free of H. defensa or harboring artificial infections with three different isolates of H. defensa. Parasitoids adapted rapidly to the presence of H. defensa in their hosts, but this adaptation was in part specific to the symbiont isolate they were evolving against and did not result in an improved infectivity on all symbiont-protected hosts. Comparisons of life-history traits among the evolved lines of parasitoids did not reveal any evidence for costs of adaptation to H. defensa in terms of correlated responses that could constrain such adaptation. These results show that parasitoids readily evolve counter-adaptations to heritable defensive symbionts of their hosts, but that different symbiont strains impose different evolutionary challenges. The symbionts thus mediate the host-parasite interaction by inducing line-by-line genetic specificity.

  18. Altered consolidation of extinction-like inhibitory learning in genotype-specific dysfunctional coping fostered by chronic stress in mice.

    PubMed

    Campus, P; Maiolati, M; Orsini, C; Cabib, S

    2016-12-15

    Genetic and stress-related factors interact to foster mental disorders, possibly through dysfunctional learning. In a previous study we reported that a temporary experience of reduced food availability increases forced swim (FS)-induced helplessness tested 14days after a first experience in mice of the standard inbred C57BL/6(B6) strain but reduces it in mice of the genetically unrelated DBA/2J (D2) strain. Because persistence of FS-induced helplessness influences adaptive coping with stress challenge and involve learning processes the present study tested whether the behavioral effects of restricted feeding involved altered consolidation of FS-related learning. First, we demonstrated that restricted feeding does not influence behavior expressed on the first FS experience, supporting a specific effect on persistence rather then development of helplessness. Second, we found that FS-induced c-fos expression in the infralimbic cortex (IL) was selectively enhanced in food-restricted (FR) B6 mice and reduced in FR D2 mice, supporting opposite alterations of consolidation processes involving this brain area. Third, we demonstrated that immediate post-FS inactivation of IL prevents 24h retention of acquired helplessness by continuously free-fed mice of both strains, indicating the requirement of a functioning IL for consolidation of FS-related learning in either mouse strain. Finally, in line with the known role of IL in consolidation of extinction memories, we found that restricted feeding selectively facilitated 24h retention of an acquired extinction in B6 mice whereas impairing it in D2 mice. These findings support the conclusion that an experience of reduced food availability strain-specifically affects persistence of newly acquired passive coping strategies by altering consolidation of extinction-like inhibitory learning. PMID:27506654

  19. Susceptibility to cytomegalovirus, parvovirus B19 and age-dependent differences in levels of rubella antibodies among pregnant women.

    PubMed

    Barlinn, Regine; Vainio, Kirsti; Samdal, Helvi Holm; Nordbø, Svein Arne; Nøkleby, Hanne; Dudman, Susanne G

    2014-05-01

    Infections caused by cytomegalovirus (CMV), parvovirus B19 (B19), and rubella can lead to serious complications in pregnant women. The aim of this study was to determine the susceptibility to CMV, B19, and rubella antibodies in pregnant women in Norway. Consecutive sera samples were collected from pregnant women in two different regions in Norway. Sera were collected from age groups; ≤19, 20-24, 25-29, 30-34, 35-39, and ≥40 years old. Of the 2,000 pregnant women tested, anti-CMV IgG was positive in 62.8% anti-parvovirus B19 IgG in 59.7% and anti-rubella IgG in 94.4%. CMV IgG susceptibility has decreased in pregnant women less than 30 years of age, from 60% in a study conducted in 1973-1974 to 37.2% in present study. There was a significant difference in CMV IgG seropositivity rate between the two regions (58.6% and 67.1%). Serum levels of rubella IgG was lowest in age group 25-29 years with a positivity rate of 91.0%. Women born before vaccination with two doses of MMR started, had both a higher positivity rate and significantly higher levels of rubella antibody titre, 96.1% and 82.2 IU/ml compared to those born after 92.9% and 41.7 IU/ml. Significantly lower anti-rubella IgG titre found in the youngest age groups highlights the need for continued antenatal screening. A considerable increase in anti-CMV-IgG seropositivity rate was observed and might be associated with higher rate of breastfeeding and a higher percentage attending day-care centres.

  20. [Seroprevalence of human parvovirus B19 in children with fever and rash in the North of Tunisia].

    PubMed

    Bouafsoun, A; Hannachi, N; Smaoui, H; Boubaker, S H; Kazdaghli, K; Laabidi, D; Boukadida, J; Kechrid, A

    2016-08-01

    The aim of the study is to evaluate the prevalence of specific antibodies anti-human parvovirus B19 (PVB19) immunoglobulin M (IgM) and IgG in children with fever and rash. This study involved 257 children aged from 7 months to 15 years with febrile rash unrelated to measles and rubella (seronegative for IgM). The sera were examined by immunoenzymatic assay. Detection of antibodies of PVB19 was done by enzyme-linked immunosorbent assay (Elisa). In our study, prevalence of immunoglobulin G (IgG) and IgM were 44 and 11.3%, respectively. Clinically, children with positive IgM serology had submitted an erythema infectiosum (13/29 cases), myocarditis (1 case), encephalitis (1 case), severe sickle cell anemia (7 cases), and immunocompromised (7 cases). The incidence rate of viral infection was 11.3%; most of the cases of PVB19 infection occurred between the months of May and August. Incidence was higher in the 10-15 years age group (21%). The prevalence of IgG antibody varied and increased with age, it rises from 38.2% in preschool children (19 months-4 years) to 53.5% in those aged between 4.5 and 15 years, reaching 58% in the 10-15 years age group. The four risk factors of PVB19 infection are: (1) those aged between 4.5 and 9 years, which is the most affected age group (P = 0.0018); (2) female gender in children aged between 19 months and 4 years (P = 0.037); (3) transfusion and (4) immune deficiency (P = 0.022 and P = 0.001, respectively). The study of the prevalence of PVB19 infection shows that viral infection is acquired early in childhood, increases with age; viral transmission is favored by the community life. Because of the widespread vaccination program against measles and rubella, the systematic search of PVB19 in front of eruptive fevers becomes important. PMID:27385036

  1. Low levels of high density lipoproteins in Turks, a population with elevated hepatic lipase. High density lipoprotein characterization and gender-specific effects of apolipoprotein e genotype.

    PubMed

    Mahley, R W; Pépin, J; Palaoğlu, K E; Malloy, M J; Kane, J P; Bersot, T P

    2000-08-01

    Turks have strikingly low levels of high density lipoprotein cholesterol (HDL-C) (10-15 mg/dL lower than those of Americans or Western Europeans) associated with elevated hepatic lipase mass and activity. Here we report that Turks have low levels of high density lipoprotein subclass 2 (HDL(2)), apoA-I-containing lipoproteins (LpA-I), and pre-beta-1 HDL and increased levels of HDL(3) and LpA-I/A-II particles (potentially an atherogenic lipid profile). The frequency distributions of HDL-C and LpA-I levels were skewed toward bimodality in Turkish women but were unimodal in Turkish men. The apoE genotype affected HDL-C and LpA-I levels in women only. In women, but not men, the varepsilon2 allele was strikingly more prevalent in those with the highest levels of HDL-C and LpA-I than in those with the lowest levels. The higher prevalence of the epsilon2 allele in these subgroups of women was not explained by plasma triglyceride levels, total cholesterol levels, age, or body mass index. The modulating effects of apoE isoforms on lipolytic hydrolysis of HDL by hepatic lipase (apoE2 preventing efficient hydrolysis) or on lipoprotein receptor binding (apoE2 interacting poorly with the low density lipoprotein receptors) may account for differences in HDL-C levels in Turkish women (the epsilon2 allele being associated with higher HDL levels). In Turkish men, who have substantially higher levels of hepatic lipase activity than women, the modulating effect of apoE may be overwhelmed. The gender-specific impact of the apoE genotype on HDL-C and LpA-I levels in association with elevated levels of hepatic lipase provides new insights into the metabolism of HDL.

  2. Trait Specific Expression Profiling of Salt Stress Responsive Genes in Diverse Rice Genotypes as Determined by Modified Significance Analysis of Microarrays

    PubMed Central

    Hossain, Mohammad R.; Bassel, George W.; Pritchard, Jeremy; Sharma, Garima P.; Ford-Lloyd, Brian V.

    2016-01-01

    Stress responsive gene expression is commonly profiled in a comparative manner involving different stress conditions or genotypes with contrasting reputation of tolerance/resistance. In contrast, this research exploited a wide natural variation in terms of taxonomy, origin and salt sensitivity in eight genotypes of rice to identify the trait specific patterns of gene expression under salt stress. Genome wide transcptomic responses were interrogated by the weighted continuous morpho-physiological trait responses using modified Significance Analysis of Microarrays. More number of genes was found to be differentially expressed under salt stressed compared to that of under unstressed conditions. Higher numbers of genes were observed to be differentially expressed for the traits shoot Na+/K+, shoot Na+, root K+, biomass and shoot Cl−, respectively. The results identified around 60 genes to be involved in Na+, K+, and anion homeostasis, transport, and transmembrane activity under stressed conditions. Gene Ontology (GO) enrichment analysis identified 1.36% (578 genes) of the entire transcriptome to be involved in the major molecular functions such as signal transduction (>150 genes), transcription factor (81 genes), and translation factor activity (62 genes) etc., under salt stress. Chromosomal mapping of the genes suggests that majority of the genes are located on chromosomes 1, 2, 3, 6, and 7. The gene network analysis showed that the transcription factors and translation initiation factors formed the major gene networks and are mostly active in nucleus, cytoplasm and mitochondria whereas the membrane and vesicle bound proteins formed a secondary network active in plasma membrane and vacuoles. The novel genes and the genes with unknown functions thus identified provide picture of a synergistic salinity response representing the potentially fundamental mechanisms that are active in the wide natural genetic background of rice and will be of greater use once their roles

  3. Role of B19' martensite deformation in stabilizing two-way shape memory behavior in NiTi

    DOE PAGES

    Benafan, O.; Padula, S. A.; Noebe, R. D.; Sisneros, T. A.; Vaidyanathan, R.

    2012-11-01

    Deformation of a B19' martensitic, polycrystallineNi49.9Ti50.1 (at. %) shape memoryalloy and its influence on the magnitude and stability of the ensuing two-way shape memory effect (TWSME) was investigated by combined ex situ mechanical experimentation and in situneutron diffraction measurements at stress and temperature. The microstructural changes (texture, lattice strains, and phase fractions) during room-temperature deformation and subsequent thermal cycling were captured and compared to the bulk macroscopic response of the alloy. With increasing uniaxial strain, it was observed that B19' martensite deformed by reorientation and detwinning with preferred selection of the (1¯50)M and (010)M variants, (201¯)B19' deformation twinning, and dislocationmore » activity. These mechanisms were indicated by changes in bulk texture from the neutron diffraction measurements. Partial reversibility of the reoriented variants and deformation twins was also captured upon load removal and thermal cycling, which after isothermal deformation to strains between 6% and 22% resulted in a strong TWSME. Consequently, TWSME functional parameters including TWSME strain, strain reduction, and transformation temperatures were characterized and it was found that prior martensite deformation to 14% strain provided the optimum condition for the TWSME, resulting in a stable two-way shape memory strain of 2.2%. Thus, isothermal deformation of martensite was found to be a quick and efficient method for creating a strong and stable TWSME in Ni₄₉.₉Ti₅₀.₁.« less

  4. Genotype-by-sex and environment-by-sex interactions influence variation in serum levels of bone-specific alkaline phosphatase in adult baboons (Papio hamadryas).

    PubMed

    Havill, L M; Mahaney, M C; Rogers, J

    2004-07-01

    While more than 77% of the people in the US with osteoporosis are women, the contributions of genotype-by-sex (G x S) and environment-by-sex interactions to sex differences in osteoporosis risk factors have not been studied. To address this issue, we conducted a statistical genetic analysis of serum concentrations of bone-specific alkaline phosphatase (Bone ALP), a highly specific marker of osteoblast function that is elevated in persons with conditions like osteoporosis characterized by excessive bone turnover or rapid bone loss. We assayed Bone ALP from 657 pedigreed baboons using a commercially available ELISA kit. Using a maximum likelihood variance decomposition approach, we treated sex as an environmental milieu in which genes influencing Bone ALP levels are expressed. We modeled the genetic covariance in Bone ALP between all relative pairs conditional on their sex so that the covariance is the product of the kinship, the genetic correlation between trait levels in the two sexes, and the genetic variances in the two sexes. Sex-specific maximum likelihood estimates (MLE) of residual heritability for Bone ALP were greater for females than for males (h2 = 0.44 vs. h2 = 0.26, respectively), but likelihood ratio tests revealed only a marginally significant difference in sex-specific genetic variances (P = 0.057). In contrast, the between-sex genetic correlation (rhoG = 0.43) was significantly less than 1.0 (P = 0.037), and the difference in sex-specific environmental variances was highly significant (P = 0.00006). We report the first evidence for G x S interactions influencing variation in an osteoporosis risk factor. The diminished between-sex genetic correlation implies that different genes influence Bone ALP levels in the two sexes. The significant differences between environmental variances suggest that unmeasured factors, including those from the internal, biological environments of the two sexes, account for a greater proportion of the Bone ALP variation in

  5. Elucidating the genotype-phenotype relationships and network perturbations of human shared and specific disease genes from an evolutionary perspective.

    PubMed

    Begum, Tina; Ghosh, Tapash Chandra

    2014-10-05

    To date, numerous studies have been attempted to determine the extent of variation in evolutionary rates between human disease and nondisease (ND) genes. In our present study, we have considered human autosomal monogenic (Mendelian) disease genes, which were classified into two groups according to the number of phenotypic defects, that is, specific disease (SPD) gene (one gene: one defect) and shared disease (SHD) gene (one gene: multiple defects). Here, we have compared the evolutionary rates of these two groups of genes, that is, SPD genes and SHD genes with respect to ND genes. We observed that the average evolutionary rates are slow in SHD group, intermediate in SPD group, and fast in ND group. Group-to-group evolutionary rate differences remain statistically significant regardless of their gene expression levels and number of defects. We demonstrated that disease genes are under strong selective constraint if they emerge through edgetic perturbation or drug-induced perturbation of the interactome network, show tissue-restricted expression, and are involved in transmembrane transport. Among all the factors, our regression analyses interestingly suggest the independent effects of 1) drug-induced perturbation and 2) the interaction term of expression breadth and transmembrane transport on protein evolutionary rates. We reasoned that the drug-induced network disruption is a combination of several edgetic perturbations and, thus, has more severe effect on gene phenotypes.

  6. Increased cardiac injury in NZB/W F1 mice received antibody against human parvovirus B19 VP1 unique region protein.

    PubMed

    Tzang, Bor-Show; Lin, Tsung-Ming; Tsai, Chun-Chou; Hsu, Jeng-Dong; Yang, Lien-Chuan; Hsu, Tsai-Ching

    2011-07-01

    Human parvovirus B19 (B19) infection has been postulated to both myocardial injury and development of systemic lupus erythematosus (SLE). However, the influence of anti-B19-VP1u antibodies on cardiac disorders in SLE is still obscure. To elucidate the effects of anti-B19-VP1u IgG in SLE, passive transfer of PBS, normal rabbit IgG or rabbit anti-B19-VP1u IgG was injected intravenously into NZB/W F1 mice, respectively. Significant expression of IL-1β, IL-6 and TNF-α were detected in NZB/W F1 mice receiving rabbit anti-B19-VP1u IgG. Markedly cardiomyocyte disarray and lymphocyte infiltration were observed in left ventricle of hearts from NZB/W F1 mice receiving rabbit anti-B19-VP1u IgG. Additionally, significant increases of matrix metalloproteinase-9 (MMP9) activity and protein expression were detected in left ventricle of hearts from NZB/W F1 mice receiving B19-VP1u IgG. Accordingly, significant increase of phosphorylated p-38 and NF-κB proteins were observed in left ventricle of hearts from NZB/W F1 mice receiving B19-VP1u IgG. However, no significant variation of cardiac atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), heart-type fatty acid-binding protein (h-FABP) and creatine kinase MB (CK-MB) were detected among all experimental groups. These findings firstly demonstrated the aggravated effects of anti-B19 VP1u IgG on cardiac injury by induction of inflammatory but not myocardial infarction-associated proteins through activation of phosphorylated p-38 and NF-κB signaling.

  7. Application of Genotyping-By-Sequencing for selection of locus-specific BAC clones to construct physical maps and identify candidate genes in Vitis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    While genotyping-by-sequencing (GBS) is widely used for linkage and association mapping, its potential for physical mapping and candidate gene identification in under-characterized species has not been fully realized. Eight half-sib Vitis families (480 progeny) were genotyped using GBS and phenotyp...

  8. Genotype-specific responses of Bromus erectus to elevated CO{sub 2} at different levels of biodiversity and endophyte infection - a field experiment

    SciTech Connect

    Steinger, T.; Groppe, K.; Schmid, B. |

    1995-06-01

    In 1994 we initiated a long-term field experiment in a calcareous grassland to study the effects of elevated CO{sub 2} on individuals, populations, and communities. Clonal replicates of 54 genotypes of the dominant grass Bromus erectus were grown in communities planted at three levels of biodiversity (5-, 12-, 31-species plots) and exposed to ambient and elevated CO{sub 2}. The same genotypes were also individually grown in tubes within the field plots. Some genotypes were infected by the endophytic fungus Epichloee typhina. Elevated CO{sub 2} had no significant effects on plant growth, however, there was large variation among genotypes in all measured characters. A significant CO{sub 2}-by-genotype interaction was found for leaf length in the competition-free tubes. Infection by the endophyte led to the abortion of all inflorescences but increased vegetative growth, especially under competitive conditions.

  9. Sex-specific plasticity and genotype × sex interactions for age and size of maturity in the sheepshead swordtail, Xiphophorus birchmanni.

    PubMed

    Boulton, K; Rosenthal, G G; Grimmer, A J; Walling, C A; Wilson, A J

    2016-03-01

    Responses to sexually antagonistic selection are thought to be constrained by the shared genetic architecture of homologous male and female traits. Accordingly, adaptive sexual dimorphism depends on mechanisms such as genotype-by-sex interaction (G×S) and sex-specific plasticity to alleviate this constraint. We tested these mechanisms in a population of Xiphophorus birchmanni (sheepshead swordtail), where the intensity of male competition is expected to mediate intersexual conflict over age and size at maturity. Combining quantitative genetics with density manipulations and analysis of sex ratio variation, we confirm that maturation traits are dimorphic and heritable, but also subject to large G×S. Although cross-sex genetic correlations are close to zero, suggesting sex-linked genes with important effects on growth and maturation are likely segregating in this population, we found less evidence of sex-specific adaptive plasticity. At high density, there was a weak trend towards later and smaller maturation in both sexes. Effects of sex ratio were stronger and putatively adaptive in males but not in females. Males delay maturation in the presence of mature rivals, resulting in larger adult size with subsequent benefit to competitive ability. However, females also delay maturation in male-biased groups, incurring a loss of reproductive lifespan without apparent benefit. Thus, in highly competitive environments, female fitness may be limited by the lack of sex-specific plasticity. More generally, assuming that selection does act antagonistically on male and female maturation traits in the wild, our results demonstrate that genetic architecture of homologous traits can ease a major constraint on the evolution of adaptive dimorphism.

  10. Egyptian genotyping of Giardia lamblia.

    PubMed

    El-Shazly, Atef M; Mowafy, Nawras; Soliman, Mohamed; El-Bendary, Mahmoud; Morsy, Ayman T A; Ramadan, Nashwa I I; Arafa, Wafaa A S

    2004-04-01

    Out of 105 patients infected with Giardia, 38 patients have Genotype I (36.19%), 13 have Genotype II (12.38%), 10 have Genotype III (9.52%), 16 have mixed Genotype infection (15.24%) and 28 with undetermined Giardia infection by PCR (26.67%). None of the control group gave positive results for Giardia in stool by PCR. So, the sensitivity of the test for detection and identification of Giardia Genotypes from the original stool samples was 73.33% and specificity was 100%. Out of 61 cases in the symptomatic group, the prevalence of Giardia Genotype I was 32.79%, Genotype II was 16.39%, Genotype III was 9.84%, mixed Genotype infection was 16.39% and undetermined Genotype was 24.59% as compared to 40.91%, 6.82%, 9.09%, 13.64% & 29.55% in the asymptomatic group respectively. There is statistically insignificant difference between both groups as regarding the prevalence of the different Giardia Genotypes. (P < or = 0.05). The use of PCR as a routine work for diagnosis of giardiasis is not accepted at least in the developing and under-developing countries due to its high cost, the high quality of technical staff and advanced laboratory equipments required for PCR performance. Its application is usually limited to research activities, the detection of water sources contamination and for the detection of a potential source of Giardia infection in epidemics.

  11. A multiplex allele-specific real-time polymerase chain reaction assay for HLA-B*13:01 genotyping in four Chinese populations.

    PubMed

    Liu, Z; Chen, G; Kang, X; Han, M; Chen, R; Chen, C; Wang, H

    2016-10-01

    Human leukocyte antigen HLA-B*13:01 is identified currently as a marker of individual susceptibility to drug-induced hypersensitivity reaction, such as dapsone-induced hypersensitivity reactions (DIHRs) and trichloroethylene-induced dermatitis. Therefore, screening for the HLA-B*13:01 allele can assist clinics in identifying patients at risk of developing DIHRs. By combining the allele-specific primers with TaqMan probes, we established a single tube, triplex real-time PCR to detect HLA-B*13:01. The reliability of this assay was validated by the comparison of genotyping results with those by sequence-based typing (SBT). With this assay, the distribution of HLA-B*13:01 in a total of 350 blood samples from four ethnic groups: Han, Tibetan, Uighur, and Buyei were determined. A 100% concordance was observed between the results with the established real-time PCR and SBT in 100 samples. The detection limit of this assay was 0.016 ng genomic DNA. The prevalence of HLA-B*13:01 carriers were 11%, 8%, 1%, and 2% in the Buyei (n = 100), Northern Han (n = 100), Tibetan (n = 100), and Uighur (n = 50) populations, respectively. The multiplex real-time PCR assay provided a fast and reliable method for accurate detection of HLA-B*13:01 allele prior to dapsone administration in clinical practice and onset of the reaction after exposure to trichloroethylene.

  12. Genotyping-by-sequencing based intra-specific genetic map refines a ''QTL-hotspot" region for drought tolerance in chickpea.

    PubMed

    Jaganathan, Deepa; Thudi, Mahendar; Kale, Sandip; Azam, Sarwar; Roorkiwal, Manish; Gaur, Pooran M; Kishor, P B Kavi; Nguyen, Henry; Sutton, Tim; Varshney, Rajeev K

    2015-04-01

    To enhance the marker density in the "QTL-hotspot" region, harboring several QTLs for drought tolerance-related traits identified on linkage group 04 (CaLG04) in chickpea recombinant inbred line (RIL) mapping population ICC 4958 × ICC 1882, a genotyping-by-sequencing approach was adopted. In total, 6.24 Gb data from ICC 4958, 5.65 Gb data from ICC 1882 and 59.03 Gb data from RILs were generated, which identified 828 novel single-nucleotide polymorphisms (SNPs) for genetic mapping. Together with these new markers, a high-density intra-specific genetic map was developed that comprised 1,007 marker loci spanning a distance of 727.29 cM. QTL analysis using the extended genetic map along with precise phenotyping data for 20 traits collected over one to seven seasons identified 49 SNP markers in the "QTL-hotspot" region. These efforts have refined the "QTL-hotspot" region to 14 cM. In total, 164 main-effect QTLs including 24 novel QTLs were identified. In addition, 49 SNPs integrated in the "QTL-hotspot" region were converted into cleaved amplified polymorphic sequence (CAPS) and derived CAPS (dCAPS) markers which can be used in marker-assisted breeding.

  13. Longevity of Genotype-Specific Immune Responses to Plasmodium falciparum Merozoite Surface Protein 1 in Kenyan Children from Regions of Different Malaria Transmission Intensity.

    PubMed

    Bowman, Natalie M; Juliano, Jonathan J; Snider, Cynthia J; Kharabora, Oksana; Meshnick, Steven R; Vulule, John; John, Chandy C; Moormann, Ann M

    2016-09-01

    Naturally acquired immunity to Plasmodium falciparum presents a changing landscape as malaria control programs and vaccine initiatives are implemented. Determining which immunologic indicators remain surrogates of past infection, as opposed to mediators of protection, led us to compare stability of immune responses across regions with divergent malaria transmission intensities. A repeat cross-sectional study of Kenyan children from a malaria-holoendemic area and an epidemic-prone area was used to examine longitudinal antibody and interferon-gamma (IFN-γ) responses to the 3D7 and FVO variants of merozoite surface protein 1 (MSP1). Antibodies to MSP1 were common in both study populations and did not significantly wane over a 21-month time period. IFN-γ responses were less frequent and rapidly disappeared in children after a prolonged period of no malaria transmission. Antibody and IFN-γ responses rarely correlated with each other; however, MSP1-specific IFN-γ response correlated with lack of concurrent P. falciparum parasitemia of the same genotype, though only statistically significantly in the malaria-holoendemic region (odds ratio = 0.31, 95% confidence interval = 0.12-0.84). This study affirms that antimalarial antibodies are informative for evaluation of history of malaria exposure within individuals, whereas cell-mediated immunity, though short lived under natural exposure conditions, might provide an assessment of recent infection and protection from parasitemia. PMID:27481054

  14. Human parvovirus B19 causes cell cycle arrest of human erythroid progenitors via deregulation of the E2F family of transcription factors

    PubMed Central

    Wan, Zhihong; Zhi, Ning; Wong, Susan; Keyvanfar, Keyvan; Liu, Delong; Raghavachari, Nalini; Munson, Peter J.; Su, Su; Malide, Daniela; Kajigaya, Sachiko; Young, Neal S.

    2010-01-01

    Human parvovirus B19 (B19V) is the only human pathogenic parvovirus. It causes a wide spectrum of human diseases, including fifth disease (erythema infectiosum) in children and pure red cell aplasia in immunocompromised patients. B19V is highly erythrotropic and preferentially replicates in erythroid progenitor cells (EPCs). Current understanding of how B19V interacts with cellular factors to regulate disease progression is limited, due to a lack of permissive cell lines and animal models. Here, we employed a recently developed primary human CD36+ EPC culture system that is highly permissive for B19V infection to identify cellular factors that lead to cell cycle arrest after B19V infection. We found that B19V exploited the E2F family of transcription factors by downregulating activating E2Fs (E2F1 to E2F3a) and upregulating repressive E2Fs (E2F4 to E2F8) in the primary CD36+ EPCs. B19V nonstructural protein 1 (NS1) was a key viral factor responsible for altering E2F1–E2F5 expression, but not E2F6–E2F8 expression. Interaction between NS1 and E2F4 or E2F5 enhanced the nuclear import of these repressive E2Fs and induced stable G2 arrest. NS1-induced G2 arrest was independent of p53 activation and increased viral replication. Downstream E2F4/E2F5 targets, which are potentially involved in the progression from G2 into M phase and erythroid differentiation, were identified by microarray analysis. These findings provide new insight into the molecular pathogenesis of B19V in highly permissive erythroid progenitors. PMID:20890043

  15. Interaction effect between handedness and CNTNAP2 polymorphism (rs7794745 genotype) on voice-specific frontotemporal activity in healthy individuals: an fMRI study

    PubMed Central

    Koeda, Michihiko; Watanabe, Atsushi; Tsuda, Kumiko; Matsumoto, Miwako; Ikeda, Yumiko; Kim, Woochan; Tateno, Amane; Naing, Banyar Than; Karibe, Hiroyuki; Shimada, Takashi; Suzuki, Hidenori; Matsuura, Masato; Okubo, Yoshiro

    2015-01-01

    Recent neuroimaging studies have demonstrated that Contactin-associated protein-like2 (CNTNAP2) polymorphisms affect left-hemispheric function of language processing in healthy individuals, but no study has investigated the influence of these polymorphisms on right-hemispheric function involved in human voice perception. Further, although recent reports suggest that determination of handedness is influenced by genetic effect, the interaction effect between handedness and CNTNAP2 polymorphisms for brain activity in human voice perception and language processing has not been revealed. We aimed to investigate the interaction effect of handedness and CNTNAP2 polymorphisms in respect to brain function for human voice perception and language processing in healthy individuals. Brain function of 108 healthy volunteers (74 right-handed and 34 non-right-handed) was examined while they were passively listening to reverse sentences (rSEN), identifiable non-vocal sounds (SND), and sentences (SEN). Full factorial design analysis was calculated by using three factors: (1) rs7794745 (A/A or A/T), (2) rs2710102 [G/G or A carrier (A/G and A/A)], and (3) voice-specific response (rSEN or SND). The main effect of rs7794745 (A/A or A/T) was significantly revealed at the right middle frontal gyrus (MFG) and bilateral superior temporal gyrus (STG). This result suggests that rs7794745 genotype affects voice-specific brain function. Furthermore, interaction effect was significantly observed among MFG-STG activations by human voice perception, rs7794745 (A/A or A/T), and handedness. These results suggest that CNTNAP2 polymorphisms could be one of the important factors in the neural development related to vocal communication and language processing in both right-handed and non-right-handed healthy individuals. PMID:25941478

  16. Monitoring human parvovirus B19 virus-like particles and antibody complexes in solution by fluorescence correlation spectroscopy.

    PubMed

    Toivola, Jouni; Michel, Patrik O; Gilbert, Leona; Lahtinen, Tomi; Marjomäki, Varpu; Hedman, Klaus; Vuento, Matti; Oker-Blom, Christian

    2004-01-01

    Fluorescence correlation spectroscopy (FCS) was used in monitoring human parvovirus B19 virus-like particle (VLP) antibody complexes from acute phase and past-immunity serum samples. The Oregon Green 488-labeled VLPs gave an average diffusion coefficient of 1.7 x 10(-7) cm2 s(-1) with an apparent hydrodynamic radius of 14 nm. After incubation of the fluorescent VLPs with an acute phase serum sample, the mobility information obtained from the fluorescence intensity fluctuation by autocorrelation analysis showed an average diffusion coefficient of 1.5 x 10(-8) cm2 s(-1), corresponding to an average radius of 157 nm. In contrast, incubation of the fluorescent VLPs with a past-immunity serum sample gave an average diffusion coefficient of 3.5 x 10(-8) cm2 s(-1) and a radius of 69 nm. A control serum devoid of B19 antibodies caused a change in the diffusion coefficient from 1.7 x 10(-7) to 1.6 x 10(-7) cm2 s(-1), which is much smaller than that observed with acute phase or past-immunity sera. Thus, VLP-antibody complexes with different diffusion coefficients could be identified for the acute phase and past-immunity sera. FCS measurement of VLP-immune complexes could be useful in distinguishing between antibodies present in acute phase or past-immunity sera as well as in titration of the VLPs.

  17. Effects of Human Parvovirus B19 and Bocavirus VP1 Unique Region on Tight Junction of Human Airway Epithelial A549 Cells

    PubMed Central

    Chiu, Chun-Ching; Shi, Ya-Fang; Yang, Jiann-Jou; Hsiao, Yuan-Chao; Tzang, Bor-Show; Hsu, Tsai-Ching

    2014-01-01

    As is widely recognized, human parvovirus B19 (B19) and human bocavirus (HBoV) are important human pathogens. Obviously, both VP1 unique region (VP1u) of B19 and HBoV exhibit the secreted phospholipase A2 (sPLA2)-like enzymatic activity and are recognized to participate in the pathogenesis of lower respiratory tract illnesses. However, exactly how, both VP1u from B19 and HBoV affect tight junction has seldom been addressed. Therefore, this study investigates how B19-VP1u and HBoV-VP1u may affect the tight junction of the airway epithelial A549 cells by examining phospholipase A2 activity and transepithelial electrical resistance (TEER) as well as performing immunoblotting analyses. Experimental results indicate that TEER is more significantly decreased in A549 cells by treatment with TNF-α (10 ng), two dosages of B19-VP1u and BoV-VP1u (400 ng and 4000 ng) or bee venom PLA2 (10 ng) than that of the control. Accordingly, more significantly increased claudin-1 and decreased occludin are detected in A549 cells by treatment with TNF-α or both dosages of HBoV-VP1u than that of the control. Additionally, more significantly decreased Na+/K+ ATPase is observed in A549 cells by treatment with TNF-α, high dosage of B19-VP1u or both dosages of BoV-VP1u than that of the control. Above findings suggest that HBoV-VP1u rather than B19 VP1u likely plays more important roles in the disruption of tight junction in the airway tract. Meanwhile, this discrepancy appears not to be associated with the secreted phospholipase A2 (sPLA2)-like enzymatic activity. PMID:25268969

  18. Effects of human Parvovirus B19 and Bocavirus VP1 unique region on tight junction of human airway epithelial A549 cells.

    PubMed

    Chiu, Chun-Ching; Shi, Ya-Fang; Yang, Jiann-Jou; Hsiao, Yuan-Chao; Tzang, Bor-Show; Hsu, Tsai-Ching

    2014-01-01

    As is widely recognized, human parvovirus B19 (B19) and human bocavirus (HBoV) are important human pathogens. Obviously, both VP1 unique region (VP1u) of B19 and HBoV exhibit the secreted phospholipase A2 (sPLA2)-like enzymatic activity and are recognized to participate in the pathogenesis of lower respiratory tract illnesses. However, exactly how, both VP1u from B19 and HBoV affect tight junction has seldom been addressed. Therefore, this study investigates how B19-VP1u and HBoV-VP1u may affect the tight junction of the airway epithelial A549 cells by examining phospholipase A2 activity and transepithelial electrical resistance (TEER) as well as performing immunoblotting analyses. Experimental results indicate that TEER is more significantly decreased in A549 cells by treatment with TNF-α (10 ng), two dosages of B19-VP1u and BoV-VP1u (400 ng and 4000 ng) or bee venom PLA2 (10 ng) than that of the control. Accordingly, more significantly increased claudin-1 and decreased occludin are detected in A549 cells by treatment with TNF-α or both dosages of HBoV-VP1u than that of the control. Additionally, more significantly decreased Na+/K+ ATPase is observed in A549 cells by treatment with TNF-α, high dosage of B19-VP1u or both dosages of BoV-VP1u than that of the control. Above findings suggest that HBoV-VP1u rather than B19 VP1u likely plays more important roles in the disruption of tight junction in the airway tract. Meanwhile, this discrepancy appears not to be associated with the secreted phospholipase A2 (sPLA2)-like enzymatic activity.

  19. Phospholipase A2 Activity-Dependent Stimulation of Ca2+ Entry by Human Parvovirus B19 Capsid Protein VP1▿

    PubMed Central

    Lupescu, Adrian; Bock, C.-Thomas; Lang, Philipp A.; Aberle, Susanne; Kaiser, Heike; Kandolf, Reinhard; Lang, Florian

    2006-01-01

    Recent reports demonstrated an association of human parvovirus B19 with inflammatory cardiomyopathy (iCMP), which is accompanied by endothelial dysfunction. As intracellular Ca2+ activity is a key regulator of cell function and participates in mechanisms leading to endothelial dysfunction, the present experiments explored the effects of the B19 capsid proteins VP1 and VP2. A secreted phospholipase A2 (PLA2)-like activity has been located in the VP1 unique region of the B19 minor capsid protein. As PLA2 has recently been shown to activate the store-operated or capacitative Ca2+ channel ICRAC, we analyzed the impact of the viral PLA2 motif on Ca2+ entry. We cloned the VP1 and VP2 genes isolated from a patient suffering from fatal B19 iCMP into eukaryotic expression vectors. We also generated a B19 replication-competent plasmid to demonstrate PLA2 activity under the control of the complete B19 genome. After the transfection of human endothelial cells (HMEC-1), cytosolic Ca2+ activity was determined by utilizing Fura-2 fluorescence. VP1 and VP2 expression did not significantly modify basal cytosolic Ca2+ activity or the decline of cytosolic Ca2+ activity following the removal of extracellular Ca2+. However, expression of VP1 and of the full-length B19 clone, but not of VP2, significantly accelerated the increase of cytosolic Ca2+ activity following the readdition of extracellular Ca2+ in the presence of thapsigargin, indicating an activation of ICRAC. The effect of VP1 was mimicked by the PLA2 product lysophosphatidylcholine and abolished by an inactivating mutation of the PLA2-encoding region of the VP1 gene. Our observations point to the activation of Ca2+ entry by VP1 PLA2 activity, an effect likely participating in the pathophysiology of B19 infection. PMID:16956939

  20. Acute parvovirus B19 infection causes nonspecificity frequently in Borrelia and less often in Salmonella and Campylobacter serology, posing a problem in diagnosis of infectious arthropathy.

    PubMed

    Tuuminen, Tamara; Hedman, Klaus; Söderlund-Venermo, Maria; Seppälä, Ilkka

    2011-01-01

    Several infectious agents may cause arthritis or arthropathy. For example, infection with Borrelia burgdorferi, the etiologic agent of Lyme disease, may in the late phase manifest as arthropathy. Infections with Campylobacter, Salmonella, or Yersinia may result in a postinfectious reactive arthritis. Acute infection with parvovirus B19 (B19V) may likewise initiate transient or chronic arthropathy. All these conditions may be clinically indistinguishable from rheumatoid arthritis. Here, we present evidence that acute B19V infection may elicit IgM antibodies that are polyspecific or cross-reactive with a variety of bacterial antigens. Their presence may lead to misdiagnosis and improper clinical management, exemplified here by two case descriptions. Further, among 33 subjects with proven recent B19V infection we found IgM enzyme immunoassay (EIA) positivity for Borrelia only; for Borrelia and Salmonella; for Borrelia and Campylobacter; and for Borrelia, Campylobacter, and Salmonella in 26 (78.7%), 1 (3%), 2 (6%), and 1 (3%), respectively; however, when examined by Borrelia LineBlot, all samples were negative. These antibodies persisted over 3 months in 4/13 (38%) patients tested. Likewise, in a retrospective comparison of the results of a diagnostic laboratory, 9/11 (82%) patients with confirmed acute B19V infection showed IgM antibody to Borrelia. However, none of 12 patients with confirmed borreliosis showed any serological evidence of acute B19V infection. Our study demonstrates that recent B19V infection can be misinterpreted as secondary borreliosis or enteropathogen-induced reactive arthritis. To obtain the correct diagnosis, we emphasize caution in interpretation of polyreactive IgM and exclusion of recent B19V infection in patients examined for infectious arthritis or arthropathy.

  1. TU-B-19A-01: Image Registration II: TG132-Quality Assurance for Image Registration

    SciTech Connect

    Brock, K; Mutic, S

    2014-06-15

    AAPM Task Group 132 was charged with a review of the current approaches and solutions for image registration in radiotherapy and to provide recommendations for quality assurance and quality control of these clinical processes. As the results of image registration are always used as the input of another process for planning or delivery, it is important for the user to understand and document the uncertainty associate with the algorithm in general and the Result of a specific registration. The recommendations of this task group, which at the time of abstract submission are currently being reviewed by the AAPM, include the following components. The user should understand the basic image registration techniques and methods of visualizing image fusion. The disclosure of basic components of the image registration by commercial vendors is critical in this respect. The physicists should perform end-to-end tests of imaging, registration, and planning/treatment systems if image registration is performed on a stand-alone system. A comprehensive commissioning process should be performed and documented by the physicist prior to clinical use of the system. As documentation is important to the safe implementation of this process, a request and report system should be integrated into the clinical workflow. Finally, a patient specific QA practice should be established for efficient evaluation of image registration results. The implementation of these recommendations will be described and illustrated during this educational session. Learning Objectives: Highlight the importance of understanding the image registration techniques used in their clinic. Describe the end-to-end tests needed for stand-alone registration systems. Illustrate a comprehensive commissioning program using both phantom data and clinical images. Describe a request and report system to ensure communication and documentation. Demonstrate an clinically-efficient patient QA practice for efficient evaluation of image

  2. Self-accommodation of B19' martensite in Ti-Ni shape memory alloys - Part II. Characteristic interface structures between habit plane variants

    NASA Astrophysics Data System (ADS)

    Nishida, M.; Okunishi, E.; Nishiura, T.; Kawano, H.; Inamura, T.; S., Ii; Hara, T.

    2012-06-01

    Four characteristic interface microstructures between habit plane variants (HPVs) in the self-accommodation morphologies of B19‧ martensite in Ti-Ni alloys have been investigated by scanning transmission electron microscopy (STEM). The straight interface of a ? B19‧ type I twin is present at interface I. The relaxation of the transformation strain at interface II is achieved by a volume reduction of the minor correspondence variants (CVs) in the relevant habit plane variants (HPVs). The relaxation of the transformation strain at interface III is mainly due to the formation of a ? B19‧ type I twin between the two major CVs. Subsequently, local strain around the tips of the minor CVs perpendicular to the interface is released by the formation of micro-twins with the ⟨011⟩B19‧ type II and/or ? B19‧ type I relation. The major and minor CVs in each HPV are alternately connected through fine variants with the ? B19‧ type I twin relation parallel to interface IV. The results are compared with macroscopic observations and the predictions of PTMC analysis.

  3. A Novel Murine Model of Parvovirus Associated Dilated Cardiomyopathy Induced by Immunization with VP1-Unique Region of Parvovirus B19

    PubMed Central

    Šimoliūnas, Egidijus; Rinkūnaitė, Ieva; Smalinskaitė, Luka; Podkopajev, Andrej; Bironaitė, Daiva; Weis, Cleo-Aron; Marx, Alexander; Bukelskienė, Virginija; Gretz, Norbert; Grabauskienė, Virginija; Labeit, Dittmar; Labeit, Siegfried

    2016-01-01

    Background. Parvovirus B19 (B19V) is a common finding in endomyocardial biopsy specimens from myocarditis and dilated cardiomyopathy patients. However, current understanding of how B19V is contributing to cardiac damage is rather limited due to the lack of appropriate mice models. In this work we demonstrate that immunization of BALB/c mice with the major immunogenic determinant of B19V located in the unique sequence of capsid protein VP1 (VP1u) is an adequate model to study B19V associated heart damage. Methods and Results. We immunized mice in the experimental group with recombinant VP1u; immunization with cardiac myosin derived peptide served as a positive reference and phosphate buffered saline served as negative control. Cardiac function and dimensions were followed echocardiographically 69 days after immunization. Progressive dilatation of left ventricle and decline of ejection fraction were observed in VP1u- and myosin-immunized mice. Histologically, severe cardiac fibrosis and accumulation of heart failure cells in lungs were observed 69 days after immunization. Transcriptomic profiling revealed ongoing cardiac remodeling and immune process in VP1u- and myosin-immunized mice. Conclusions. Immunization of BALB/c mice with VP1u induces dilated cardiomyopathy in BALB/c mice and it could be used as a model to study clinically relevant B19V associated cardiac damage. PMID:27812527

  4. Compressed Genotyping

    PubMed Central

    Erlich, Yaniv; Gordon, Assaf; Brand, Michael; Hannon, Gregory J.; Mitra, Partha P.

    2011-01-01

    Over the past three decades we have steadily increased our knowledge on the genetic basis of many severe disorders. Nevertheless, there are still great challenges in applying this knowledge routinely in the clinic, mainly due to the relatively tedious and expensive process of genotyping. Since the genetic variations that underlie the disorders are relatively rare in the population, they can be thought of as a sparse signal. Using methods and ideas from compressed sensing and group testing, we have developed a cost-effective genotyping protocol to detect carriers for severe genetic disorders. In particular, we have adapted our scheme to a recently developed class of high throughput DNA sequencing technologies. The mathematical framework presented here has some important distinctions from the ’traditional’ compressed sensing and group testing frameworks in order to address biological and technical constraints of our setting. PMID:21451737

  5. MO-B-19A-01: MOC: A How-To Guide

    SciTech Connect

    Ibbott, G; Seibert, J; Allison, J; Frey, G

    2014-06-15

    Medical physicists who were certified in 2002 or later, as well as those who become certified in the future, are enrolled in Maintenance of Certification. Many physicists with life-time certificates have voluntarily enrolled in MOC, as have physicists who volunteer their time to participate in the ABR exam development and administration processes. MOC consists of four components: Part 1, Professional standing; Part 2, Lifelong learning and self-assessment; Part 3, Cognitive expertise; and Part 4, Practice quality improvement. These four components together evaluate six competencies: Medical knowledge, patient care and procedural skills, interpersonal and communication skills, professionalism, practice-based learning and improvement, and systems-based practice. Parts 1, 2, and 3 of MOC are fairly straightforward, although many participants have questions about the process for attesting to professional standing, the opportunities for obtaining self-assessed continuing education, and the timing of the cognitive exam. MOC participants also have questions about Part 4, Practice Quality Improvement. PQI projects are powerful tools for improving the quality and safety of the environments in which we practice medical physics. In the current version of MOC known as “Continuous Certification” a medical physicist must have completed a PQI project within the previous three years, at the time of the ABR's annual look-back each March. For the first “full” annual look-back in March 2016, diplomates will be given an additional year, so that a PQI project completed in 2012, 2013, 2014, or 2015 will fulfill this requirement. Each component of MOC will be addressed, and the specifics of interest to medical physicists will be discussed. Learning Objectives: Understand the four components and six competencies evaluated by MOC. Become familiar with the annual requirements of Continuous Certification. Learn about opportunities for Practice Quality Improvement projects. Understand

  6. SSR genotyping.

    PubMed

    Mason, Annaliese S

    2015-01-01

    SSR genotyping involves the use of simple sequence repeats (SSRs) as DNA markers. SSRs, also called microsatellites, are a type of repetitive DNA sequence ubiquitous in most plant genomes. SSRs contain repeats of a motif sequence 1-6 bp in length. Due to this structure SSRs frequently undergo mutations, mainly due to DNA polymerase errors, which involve the addition or subtraction of a repeat unit. Hence, SSR sequences are highly polymorphic and may be readily used for detection of allelic variation within populations. SSRs are present within both genic and nongenic regions and are occasionally transcribed, and hence may be identified in expressed sequence tags (ESTs) as well as more commonly in nongenic DNA sequences. SSR genotyping involves the design of DNA-based primers to amplify SSR sequences from extracted genomic DNA, followed by amplification of the SSR repeat region using polymerase chain reaction, and subsequent visualization of the resulting DNA products, usually using gel electrophoresis. These procedures are described in this chapter. SSRs have been one of the most favored molecular markers for plant genotyping in the last 20 years due to their high levels of polymorphism, wide distribution across most plant genomes, and ease of use and will continue to be a useful tool in many species for years to come.

  7. TORCH Infections. Toxoplasmosis, Other (syphilis, varicella-zoster, parvovirus B19), Rubella, Cytomegalovirus (CMV), and Herpes infections.

    PubMed

    Stegmann, Barbara J; Carey, J Christopher

    2002-08-01

    Perinatal infections account for 2% to 3% of all congenital anomalies. TORCH, which includes Toxoplasmosis, Other (syphilis, varicella-zoster, parvovirus B19), Rubella, Cytomegalovirus (CMV), and Herpes infections, are some of the most common infections associated with congenital anomalies. Most of the TORCH infections cause mild maternal morbidity, but have serious fetal consequences, and treatment of maternal infection frequently has no impact on fetal outcome. Therefore, recognition of maternal disease and fetal monitoring once disease is recognized are important for all clinicians. Knowledge of these diseases will help the clinician appropriately counsel mothers on preventive measures to avoid these infections, and will aid in counseling parents on the potential for adverse fetal outcomes when these infections are present. PMID:12150751

  8. The First High-Density Genetic Map Construction in Tree Peony (Paeonia Sect. Moutan) using Genotyping by Specific-Locus Amplified Fragment Sequencing

    PubMed Central

    Cai, Changfu; Cheng, Fang-Yun; Wu, Jing; Zhong, Yuan; Liu, Gaixiu

    2015-01-01

    Genetic linkage maps, permitting the elucidation of genome structure, are one of most powerful genomic tools to accelerate marker-assisted breeding. However, due to a lack of sufficient user-friendly molecular markers, no genetic linkage map has been developed for tree peonies (Paeonia Sect. Moutan), a group of important horticultural plants worldwide. Specific-locus amplified fragment sequencing (SLAF-seq) is a recent molecular marker development technology that enable the large-scale discovery and genotyping of sequence-based marker in genome-wide. In this study, we performed SLAF sequencing of an F1 population, derived from the cross P. ostti ‘FenDanBai’ × P. × suffruticosa ‘HongQiao’, to identify sufficient high-quality markers for the construction of high-density genetic linkage map in tree peonies. After SLAF sequencing, a total of 78 Gb sequencing data and 285,403,225 pair-end reads were generated. We detected 309,198 high-quality SLAFs from these data, of which 85,124 (27.5%) were polymorphic. Subsequently, 3518 of the polymorphic markers, which were successfully encoded in to Mendelian segregation types, and were in conformity with the criteria of high-quality markers, were defined as effective markers and used for genetic linkage mapping. Finally, we constructed an integrated genetic map, which comprised 1189 markers on the five linkage groups, and spanned 920.699 centiMorgans (cM) with an average inter-marker distance of 0.774 cM. There were 1115 ‘SNP-only’ markers, 18 ‘InDel-only’ markers, and 56 ‘SNP&InDel’ markers on the map. Among these markers, 450 (37.85%) showed significant segregation distortion (P < 0.05). In conclusion, this investigation reported the first large-scale marker development and high-density linkage map construction for tree peony. The results of this study will serve as a solid foundation not only for marker-assisted breeding, but also for genome sequence assembly for tree peony. PMID:26010095

  9. The First High-Density Genetic Map Construction in Tree Peony (Paeonia Sect. Moutan) using Genotyping by Specific-Locus Amplified Fragment Sequencing.

    PubMed

    Cai, Changfu; Cheng, Fang-Yun; Wu, Jing; Zhong, Yuan; Liu, Gaixiu

    2015-01-01

    Genetic linkage maps, permitting the elucidation of genome structure, are one of most powerful genomic tools to accelerate marker-assisted breeding. However, due to a lack of sufficient user-friendly molecular markers, no genetic linkage map has been developed for tree peonies (Paeonia Sect. Moutan), a group of important horticultural plants worldwide. Specific-locus amplified fragment sequencing (SLAF-seq) is a recent molecular marker development technology that enable the large-scale discovery and genotyping of sequence-based marker in genome-wide. In this study, we performed SLAF sequencing of an F1 population, derived from the cross P. ostti 'FenDanBai' × P. × suffruticosa 'HongQiao', to identify sufficient high-quality markers for the construction of high-density genetic linkage map in tree peonies. After SLAF sequencing, a total of 78 Gb sequencing data and 285,403,225 pair-end reads were generated. We detected 309,198 high-quality SLAFs from these data, of which 85,124 (27.5%) were polymorphic. Subsequently, 3518 of the polymorphic markers, which were successfully encoded in to Mendelian segregation types, and were in conformity with the criteria of high-quality markers, were defined as effective markers and used for genetic linkage mapping. Finally, we constructed an integrated genetic map, which comprised 1189 markers on the five linkage groups, and spanned 920.699 centiMorgans (cM) with an average inter-marker distance of 0.774 cM. There were 1115 'SNP-only' markers, 18 'InDel-only' markers, and 56 'SNP&InDel' markers on the map. Among these markers, 450 (37.85%) showed significant segregation distortion (P < 0.05). In conclusion, this investigation reported the first large-scale marker development and high-density linkage map construction for tree peony. The results of this study will serve as a solid foundation not only for marker-assisted breeding, but also for genome sequence assembly for tree peony.

  10. Seroprevalence study in forestry workers from eastern Germany using novel genotype 3- and rat hepatitis E virus-specific immunoglobulin G ELISAs.

    PubMed

    Dremsek, Paul; Wenzel, Jürgen J; Johne, Reimar; Ziller, Mario; Hofmann, Jörg; Groschup, Martin H; Werdermann, Sandra; Mohn, Ulrich; Dorn, Silvia; Motz, Manfred; Mertens, Marc; Jilg, Wolfgang; Ulrich, Rainer G

    2012-05-01

    Hepatitis E virus (HEV) is the causative agent of an acute self-limiting hepatitis in humans. In industrialized countries, autochthonous cases are linked to zoonotic transmission from domestic pigs, wild boar and red deer. The main route of human infection presumably is consumption of contaminated meat. Farmers, slaughterers and veterinarians are expected to be risk groups as they work close to potentially infected animals. In this study, we tested four Escherichia coli-expressed segments of the capsid protein (CP) of a German wild boar-derived HEV genotype 3 strain for their diagnostic value in an indirect immunoglobulin G (IgG) ELISA. In an initial validation experiment, a carboxy-terminal CP segment spanning amino acid (aa) residues 326-608 outperformed the other segments harbouring aa residues 112-608, 326-660 and 112-335. Based on this segment, an indirect ELISA for detection of anti-HEV IgG antibodies in human sera was established and validated using a commercial line immunoassay as reference assay. A total of 563 sera from forestry workers of all forestry offices of Brandenburg, eastern Germany and 301 sera of blood donors from eastern Germany were surveyed using these assays. The commercial test revealed seroprevalence rates of 11% for blood donors and 18% for forestry workers. These rates are in line with data obtained by the in-house test (12 and 21%). Hence, the in-house test performed strikingly similar to the commercial test (sensitivity 0.9318, specificity 0.9542). An initial screening of forestry worker and blood donor sera with a corresponding CP segment of the recently discovered Norway rat-associated HEV revealed several strong positive sera exclusively in the forestry worker panel. Future investigations have to prove the performance of this novel IgG ELISA in large-scale seroepidemiological studies. In addition, the observed elevated seroprevalence in a forestry worker group has to be confirmed by studies on groups of forestry workers from other

  11. Melanoma cultures show different susceptibility towards E1A-, E1B-19 kDa- and fiber-modified replication-competent adenoviruses.

    PubMed

    Schmitz, M; Graf, C; Gut, T; Sirena, D; Peter, I; Dummer, R; Greber, U F; Hemmi, S

    2006-06-01

    Replicating adenovirus (Ad) vectors with tumour tissue specificity hold great promise for treatment of cancer. We have recently constructed a conditionally replicating Ad5 AdDeltaEP-TETP inducing tumour regression in a xenograft mouse model. For further improvement of this vector, we introduced four genetic modifications and analysed the viral cytotoxicity in a large panel of melanoma cell lines and patient-derived melanoma cells. (1) The antiapoptotic gene E1B-19 kDa (Delta19 mutant) was deleted increasing the cytolytic activity in 18 of 21 melanoma cells. (2) Introduction of the E1A 122-129 deletion (Delta24 mutant), suggested to attenuate viral replication in cell cycle-arrested cells, did not abrogate this activity and increased the cytolytic activity in two of 21 melanoma cells. (3) We inserted an RGD sequence into the fiber to extend viral tropism to alphav integrin-expressing cells, and (4) swapped the fiber with the Ad35 fiber (F35) enhancing the tropism to malignant melanoma cells expressing CD46. The RGD-fiber modification strongly increased cytolysis in all of the 11 CAR-low melanoma cells. The F35 fiber-chimeric vector boosted the cytotoxicity in nine of 11 cells. Our results show that rational engineering additively enhances the cytolytic potential of Ad vectors, a prerequisite for the development of patient-customized viral therapies. PMID:16482201

  12. Melanoma cultures show different susceptibility towards E1A-, E1B-19 kDa- and fiber-modified replication-competent adenoviruses.

    PubMed

    Schmitz, M; Graf, C; Gut, T; Sirena, D; Peter, I; Dummer, R; Greber, U F; Hemmi, S

    2006-06-01

    Replicating adenovirus (Ad) vectors with tumour tissue specificity hold great promise for treatment of cancer. We have recently constructed a conditionally replicating Ad5 AdDeltaEP-TETP inducing tumour regression in a xenograft mouse model. For further improvement of this vector, we introduced four genetic modifications and analysed the viral cytotoxicity in a large panel of melanoma cell lines and patient-derived melanoma cells. (1) The antiapoptotic gene E1B-19 kDa (Delta19 mutant) was deleted increasing the cytolytic activity in 18 of 21 melanoma cells. (2) Introduction of the E1A 122-129 deletion (Delta24 mutant), suggested to attenuate viral replication in cell cycle-arrested cells, did not abrogate this activity and increased the cytolytic activity in two of 21 melanoma cells. (3) We inserted an RGD sequence into the fiber to extend viral tropism to alphav integrin-expressing cells, and (4) swapped the fiber with the Ad35 fiber (F35) enhancing the tropism to malignant melanoma cells expressing CD46. The RGD-fiber modification strongly increased cytolysis in all of the 11 CAR-low melanoma cells. The F35 fiber-chimeric vector boosted the cytotoxicity in nine of 11 cells. Our results show that rational engineering additively enhances the cytolytic potential of Ad vectors, a prerequisite for the development of patient-customized viral therapies.

  13. Protein-Peptide Arrays for Detection of Specific Anti-Hepatitis D Virus (HDV) Genotype 1, 6, and 8 Antibodies among HDV-Infected Patients by Surface Plasmon Resonance Imaging

    PubMed Central

    Villiers, Marie-Bernadette; Cortay, Jean-Claude; Cortès, Sandra; Bloquel, Bénédicte; Brichler, Ségolène; Brakha, Carine; Kay, Alan; Falah, Nisrine; Zoulim, Fabien; Marquette, Christophe

    2015-01-01

    Liver diseases linked to hepatitis B-hepatitis D virus co- or superinfections are more severe than those during hepatitis B virus (HBV) monoinfection. The diagnosis of hepatitis D virus (HDV) infection therefore remains crucial in monitoring patients but is often overlooked. To integrate HDV markers into high-throughput viral hepatitis diagnostics, we studied the binding of anti-HDV antibodies (Abs) using surface plasmon resonance imaging (SPRi). We focused on the ubiquitous HDV genotype 1 (HDV1) and the more uncommon African-HDV6 and HDV8 genotypes to define an array with recombinant proteins or peptides. Full-length and truncated small hepatitis D antigen (S-HDAg) recombinant proteins of HDV genotype 1 (HDV1) and 11 HDV peptides of HDV1, 6, and 8, representing various portions of the delta antigen were grafted onto biochips, allowing SPRi measurements to be made. Sixteen to 17 serum samples from patients infected with different HDV genotypes were injected onto protein and peptide chips. In all, Abs against HDV proteins and/or peptides were detected in 16 out of 17 infected patients (94.12%), although the amplitude of the SPR signal varied. The amino-terminal part of the protein was poorly immunogenic, while epitope 65-80, exposed on the viral ribonucleoprotein, may be immunodominant, as 9 patient samples led to a specific SPR signal on peptide 65 type 1 (65#1), independently of the infecting genotype. In this pilot study, we confirmed that HDV infection screening based on the reactivity of patient Abs against carefully chosen HDV peptides and/or proteins can be included in a syndrome-based viral hepatitis diagnostic assay. The preliminary results indicated that SPRi studying direct physical HDAg–anti-HDV Ab interactions was more convenient using linear peptide epitopes than full-length S-HDAg proteins, due to the regeneration process, and may represent an innovative approach for a hepatitis syndrome–viral etiology-exploring array. PMID:25631795

  14. Genotypes of p53 codon 72 correlate with age at onset of type 1 diabetes in a sex-specific manner.

    PubMed

    Bitti, Maria Luisa Manca; Saccucci, Patrizia; Capasso, Francesca; Piccinini, Simona; Angelini, Federica; Rapini, Novella; Porcari, Marta; Arcano, Susanna; Petrelli, Arianna; Del Duca, Elisabetta; Bottini, Egidio; Gloria-Bottini, Fulvia

    2011-01-01

    In type 1 diabetes mellitus (T1D) p53 pathways are up-regulated and there is an increased susceptibility to apoptosis. The hypothesis is that p53 codon 72 polymorphism could be associated with T1D. A total of 286 children with T1D and a control sample of 730 subjects were studied. p53 codon 72 polymorphism was analysed by polymerase chain reaction. A large increase of p53 *Arg/*Arg was observed in T1D patients with age at onset < 6 years. A strong linear correlation between *Arg/*Arg genotype and age at onset was observed in females. The involvement of the *Arg/*Arg genotype in apoptosis suggests that during the autoimmune process leading to T1D, genetic factors that favor apoptosis may contribute to the onset of overt disease. PMID:21932578

  15. Inactivated porcine reproductive and respiratory syndrome virus vaccine adjuvanted with Montanide™ Gel 01 ST elicits virus-specific cross-protective inter-genotypic response in piglets.

    PubMed

    Tabynov, Kairat; Sansyzbay, Abylay; Tulemissova, Zhanara; Tabynov, Kaissar; Dhakal, Santosh; Samoltyrova, Aigul; Renukaradhya, Gourapura J; Mambetaliyev, Muratbay

    2016-08-30

    The efficacy of a novel BEI-inactivated porcine reproductive and respiratory syndrome virus (PRRSV) candidate vaccine in pigs, developed at RIBSP Republic of Kazakhstan and delivered with an adjuvant Montanide™ Gel 01 ST (D/KV/ADJ) was compared with a commercial killed PRRSV vaccine (NVDC-JXA1, C/KV/ADJ) used widely in swine herds of the Republic of Kazakhstan. Clinical parameters (body temperature and respiratory disease scores), virological and immunological profiles [ELISA and virus neutralizing (VN) antibody titers], macroscopic lung lesions and viral load in the lungs (quantitative real-time PCR and cell culture assay) were assessed in vaccinated and both genotype 1 and 2 PRRSV challenged pigs. Our results showed that the commercial vaccine failed to protect pigs adequately against the clinical disease, viremia and lung lesions caused by the challenged field isolates, Kazakh strains of PRRSV type 1 and type 2 genotypes. In contrast, clinical protection, absence of viremia and lung lesions in D/KV/ADJ vaccinated pigs was associated with generation of VN antibodies in both homologous vaccine strain LKZ/2010 (PRRSV type 2) and a heterogeneous type 1 PRRSV strain (CM/08) challenged pigs. Thus, our data indicated the induction of cross-protective VN antibodies by D/KV/ADJ vaccine, and importantly demonstrated that an inactivated PRRSV vaccine could also induce cross-protective response across the viral genotype. PMID:27527768

  16. Collaborative study for establishment of a European Pharmacopoei Biological Reference Preparation (BRP) for B19 virus DNA testing of plasma pools by nucleic acid amplification technique.

    PubMed

    Nübling, C M; Daas, A; Buchheit, K H

    2004-01-01

    The goal of the collaborative study was to calibrate the B19 DNA content of a candidate Biological Reference Preparation (BRP) that is intended to be used for the validation of the analytical procedure, as threshold control and/or as quantitative reference material in the Nucleic Acid Amplification Technique (NAT) test of plasma pools for detection of B19 contamination. The candidate BRP was calibrated against the 1st International Standard for B19 DNA NAT assays. According to the European Pharmacopoeia monograph Human anti-D immunoglobulin, the threshold control needs to have a titre of 10( 4) IU/ml of B19 virus DNA. The lyophilised candidate BRP was prepared from 0.5 ml aliquots of a plasma pool spiked with B19 virus. The B19 virus originated from a "B19 virus window phase" blood donation (anti-B19 negative, B19-DNA high titre positive) and was diluted in a plasma pool tested negative by both serological and NAT assays for Hepatitis B Virus, Hepatitis C Virus and Human Immunodeficiency Virus 1 to obtain a B19-DNA concentration level in the range of 10( 6) copies/ml. The residual water content of the lyophilised candidate BRP was determined as 0.98 +/- 0.65% (mean +/- relative standard deviation). Sixteen laboratories (Official Medicine Control Laboratories, manufacturers of plasma derivatives, NAT test laboratories and NAT kit manufacturers) from nine countries participated. Participants were requested to test the candidate BRP and the International Standard (99/800) in four independent test runs on different days using their in-house qualitative and/or quantitative NAT methods. Sixteen laboratories reported results. Thirteen laboratories reported results from qualitative assays and 5 laboratories reported results from quantitative assays. Two laboratories reported results from both types of assay. For the qualitative assays a weighted combined potency of 5.64 log( 10) IU/ml with 95 per cent confidence limits of +/- 0.17 log( 10) which corresponds to 67 to 150

  17. Collaborative study for establishment of a European Pharmacopoei Biological Reference Preparation (BRP) for B19 virus DNA testing of plasma pools by nucleic acid amplification technique.

    PubMed

    Nübling, C M; Daas, A; Buchheit, K H

    2004-01-01

    The goal of the collaborative study was to calibrate the B19 DNA content of a candidate Biological Reference Preparation (BRP) that is intended to be used for the validation of the analytical procedure, as threshold control and/or as quantitative reference material in the Nucleic Acid Amplification Technique (NAT) test of plasma pools for detection of B19 contamination. The candidate BRP was calibrated against the 1st International Standard for B19 DNA NAT assays. According to the European Pharmacopoeia monograph Human anti-D immunoglobulin, the threshold control needs to have a titre of 10( 4) IU/ml of B19 virus DNA. The lyophilised candidate BRP was prepared from 0.5 ml aliquots of a plasma pool spiked with B19 virus. The B19 virus originated from a "B19 virus window phase" blood donation (anti-B19 negative, B19-DNA high titre positive) and was diluted in a plasma pool tested negative by both serological and NAT assays for Hepatitis B Virus, Hepatitis C Virus and Human Immunodeficiency Virus 1 to obtain a B19-DNA concentration level in the range of 10( 6) copies/ml. The residual water content of the lyophilised candidate BRP was determined as 0.98 +/- 0.65% (mean +/- relative standard deviation). Sixteen laboratories (Official Medicine Control Laboratories, manufacturers of plasma derivatives, NAT test laboratories and NAT kit manufacturers) from nine countries participated. Participants were requested to test the candidate BRP and the International Standard (99/800) in four independent test runs on different days using their in-house qualitative and/or quantitative NAT methods. Sixteen laboratories reported results. Thirteen laboratories reported results from qualitative assays and 5 laboratories reported results from quantitative assays. Two laboratories reported results from both types of assay. For the qualitative assays a weighted combined potency of 5.64 log( 10) IU/ml with 95 per cent confidence limits of +/- 0.17 log( 10) which corresponds to 67 to 150

  18. Response of a Habitat-Forming Marine Plant to a Simulated Warming Event Is Delayed, Genotype Specific, and Varies with Phenology.

    PubMed

    Reynolds, Laura K; DuBois, Katherine; Abbott, Jessica M; Williams, Susan L; Stachowicz, John J

    2016-01-01

    Growing evidence shows that increasing global temperature causes population declines and latitudinal shifts in geographical distribution for plants living near their thermal limits. Yet, even populations living well within established thermal limits of a species may suffer as the frequency and intensity of warming events increase with climate change. Adaptive response to this stress at the population level depends on the presence of genetic variation in thermal tolerance in the populations in question, yet few data exist to evaluate this. In this study, we examined the immediate effects of a moderate warming event of 4.5°C lasting 5 weeks and the legacy effects after a 5 week recovery on different genotypes of the marine plant Zostera marina (eelgrass). We conducted the experiment in Bodega Bay, CA USA, where average summer water temperatures are 14-15°C, but extended warming periods of 17-18°C occur episodically. Experimental warming increased shoot production by 14% compared to controls held at ambient temperature. However, after returning temperature to ambient levels, we found strongly negative, delayed effects of warming on production: shoot production declined by 27% and total biomass decreased by 50% relative to individuals that had not been warmed. While all genotypes' production decreased in the recovery phase, genotypes that grew the most rapidly under benign thermal conditions (control) were the most susceptible to the detrimental effects of warming. This suggests a potential tradeoff in relative performance at normal vs. elevated temperatures. Modest short-term increases in water temperature have potentially prolonged negative effects within the species' thermal envelope, but genetic variation within these populations may allow for population persistence and adaptation. Further, intraspecific variation in phenology can result in maintenance of population diversity and lead to enhanced production in diverse stands given sufficient frequency of warming

  19. Response of a Habitat-Forming Marine Plant to a Simulated Warming Event Is Delayed, Genotype Specific, and Varies with Phenology.

    PubMed

    Reynolds, Laura K; DuBois, Katherine; Abbott, Jessica M; Williams, Susan L; Stachowicz, John J

    2016-01-01

    Growing evidence shows that increasing global temperature causes population declines and latitudinal shifts in geographical distribution for plants living near their thermal limits. Yet, even populations living well within established thermal limits of a species may suffer as the frequency and intensity of warming events increase with climate change. Adaptive response to this stress at the population level depends on the presence of genetic variation in thermal tolerance in the populations in question, yet few data exist to evaluate this. In this study, we examined the immediate effects of a moderate warming event of 4.5°C lasting 5 weeks and the legacy effects after a 5 week recovery on different genotypes of the marine plant Zostera marina (eelgrass). We conducted the experiment in Bodega Bay, CA USA, where average summer water temperatures are 14-15°C, but extended warming periods of 17-18°C occur episodically. Experimental warming increased shoot production by 14% compared to controls held at ambient temperature. However, after returning temperature to ambient levels, we found strongly negative, delayed effects of warming on production: shoot production declined by 27% and total biomass decreased by 50% relative to individuals that had not been warmed. While all genotypes' production decreased in the recovery phase, genotypes that grew the most rapidly under benign thermal conditions (control) were the most susceptible to the detrimental effects of warming. This suggests a potential tradeoff in relative performance at normal vs. elevated temperatures. Modest short-term increases in water temperature have potentially prolonged negative effects within the species' thermal envelope, but genetic variation within these populations may allow for population persistence and adaptation. Further, intraspecific variation in phenology can result in maintenance of population diversity and lead to enhanced production in diverse stands given sufficient frequency of warming

  20. Somatic cell count and milk neutrophil viability of dairy heifers with specific CXCR1 genotypes following experimental intramammary infection with Staphylococcus chromogenes originating from milk.

    PubMed

    Verbeke, Joren; Piccart, Kristine; Piepers, Sofie; Van Poucke, Mario; Peelman, Luc; De Visscher, Anneleen; De Vliegher, Sarne

    2015-06-01

    Previous observational studies suggest an association between polymorphism c.980A>G in the CXCR1 gene, encoding the chemokine (C-X-C motif) receptor 1, and the innate immunity and infection status of the mammary gland. Mammary glands of eight Holstein heifers were experimentally infected with a Staphylococcus chromogenes isolate originating from a chronic intramammary infection (IMI) to study differences between CXCR1 genotypes c.980AG and c.980GG. Quarters from heifers with genotypes c.980AG and c.980GG developed subclinical mastitis but showed differences in the early response at 6-18 h post challenge. Bacterial count at 18 h post challenge tended to be higher in quarters from c.980AG heifers compared to c.980GG heifers. Somatic cell count (SCC) was higher at 6 h post challenge and tended to be higher at 9 h post challenge in c.980AG heifers compared to c.980GG heifers. Milk production decreased similarly. Milk neutrophils of c.980AG heifers showed more apoptosis at 9 h post challenge and tended to show more necrosis at 6, 9 and 12 h post challenge than c.980GG heifers. Differences were less pronounced in the later stage (>18 h) of infection. The results demonstrate that CXCR1 polymorphism can influence SCC and milk neutrophil viability following experimental IMI. PMID:25933826

  1. [Seroprevalence of rubella virus, varicella zoster virus, cytomegalovirus and parvovirus B19 among pregnant women in the Sousse region, Tunisia].

    PubMed

    Hannachi, N; Marzouk, M; Harrabi, I; Ferjani, A; Ksouri, Z; Ghannem, H; Khairi, H; Hidar, S; Boukadida, J

    2011-02-01

    The aim of the study is to evaluate seroprevalence of rubella virus (RV), cytomegalovirus (CMV), varicella zoster virus (VZV), and parvovirus B19 (PB19) in 404 Tunisian pregnant women, and to determine reliability of maternal past history of eruption. Sociodemographic characteristics, risk factors, and past history of eruption were collected through a questionnaire. Serologic tests were performed using enzyme immunoassays. Risk factors were analyzed using univariate and multivariate logistic regression models. Seroprevalences were 79.7% for rubella, 96.3% for CMV, 80.9% for VZV, and 76.2% for PB19. In multivariate analysis, the number of persons per room (> 2) in the house during childhood was associated with CMV infection (P = 0.004), irregular professional husband's activity was correlated with VZV infection (P = 0.04), and an age of more than 30 years was associated with PB19 infection (P = 0.02). History of rubella, varicella, and PB19 infection was unknown for, respectively, 55.8%, 20%, and 100% of women. False history of rubella and varicella were found for 7.4% and 15% of women, respectively. The positive and negative predictive values (PPV and NPV) of rubella history were, respectively, 92.6% and 17.2%, and were, respectively, 84.9% and 20.9% for varicella history. Susceptibility to RV, VZV, and PB19 infection remains high in pregnancy in our population. Preventive strategies against congenital rubella must be reinforced. Vaccination against VZV should be considered in seronegative women. Systemic CMV screening is not warranted in our country where high immunity is acquired probably in childhood. Since maternal history of eruption is not reliable, we recommend serologic testing to determine immune status of women. PMID:21243459

  2. PRDM1 expression via human parvovirus B19 infection plays a role in the pathogenesis of Hashimoto thyroiditis.

    PubMed

    Wang, Lu; Zhang, Wei-Ping; Yao, Li; Zhang, Wei; Zhu, Jin; Zhang, Wei-Chen; Zhang, Yue-Hua; Wang, Zhe; Yan, Qing-Guo; Guo, Ying; Fan, Lin-Ni; Liu, Yi-Xiong; Huang, Gao-Sheng

    2015-12-01

    Ectopic lymphoid follicle infiltration is a key event in Hashimoto thyroiditis (HT). Positive regulatory domain zinc finger protein 1 (PRDM1), which is induced by antigen stimulation, can regulate all lymphocyte lineages. Several groups independently demonstrated that human parvovirus B19 (PVB19) is closely associated with HT. Hence, we determined whether PRDM1 is expressed in HT thyroid tissue and whether there is any correlation between PRDM1 expression and PVB19 in the pathogenesis of HT. We detected PRDM1 expression in HT (n = 86), normal thyroid tissue (n = 30), and nontoxic nodular goiter (n = 20) samples using immunohistochemistry. We also detected PVB19 protein in HT samples in a double-blind manner and analyzed the correlation between the 2 proteins using immunofluorescence confocal detection and coimmunoprecipitation. Furthermore, we detected changes of the expression levels of PRDM1 and PVB19 in transfected primary thyroid follicular epithelial cells using real-time quantitative polymerase chain reaction. We found that PRDM1 protein is significantly highly expressed in the injured follicular epithelial cells in HT (83/86 cases) than in normal thyroid cells (0/30 cases) or in nontoxic nodular goiter cells (0/20 cases) (P < .001). In HT, the PRDM1 expression pattern was the same as that of PVB19, whereas PRDM1 and PVB19 were coexistent in the involved epithelial cells. Statistical analysis showed a significant correlation between PRDM1 and PVB19 (P < .001). In addition, primary thyroid epithelial cells also showed PRDM1 up-regulation after PVB19 NS1 transfection. Our findings suggest a previously unrecognized role of PRDM1 and PVB19 in the pathogenesis of HT.

  3. Activation of synoviocytes by the secreted phospholipase A2 motif in the VP1-unique region of parvovirus B19 minor capsid protein.

    PubMed

    Lu, Jun; Zhi, Ning; Wong, Susan; Brown, Kevin E

    2006-02-15

    Parvovirus B19 infection in adults is often associated with acute symmetrical polyarthropathy, but the mechanism is unknown. Recently, a secreted phospholipase A(2) (sPLA(2)) motif was identified in the VP1-unique region (VP1u) of the B19 minor capsid protein. To investigate the role of this motif, we expressed VP1u with and without point mutations in the critical amino acids of sPLA(2). Although high concentrations of B19 did not infect human fibroblast-like synoviocytes (HFLSs), there was a >3-fold increase in synoviocyte migration that could be blocked by phospholipase inhibitors. Recombinant proteins with intact VP1u demonstrated sPLA(2) activity and induced cell migration, whereas proteins with mutated VP1u were nonfunctional in both assays. The incubation of HFLSs with proteins that had intact VP1u, but not with proteins with mutated VP1u, increased the production of prostaglandin E(2) >100-fold. Expression of cyclooxygenase (COX)-2 mRNA transcripts, as determined by real-time reverse-transcription polymerase chain reaction, and COX-2 protein expression were both significantly increased after incubation with protein that had intact VP1u. Proteins with VP1u in noninfectious B19 may participate in the inflammatory response in the synovial compartment.

  4. Molecular characterization of the basidiomycete isolate Nematoloma frowardii b19 and its manganese peroxidase places the fungus in the corticioid genus Phlebia.

    PubMed

    Hildén, Kristiina S; Bortfeldt, Ralf; Hofrichter, Martin; Hatakka, Annele; Lundell, Taina K

    2008-08-01

    The basidiomycete isolate b19, originally identified by morphological characteristics of the fruiting body as Nematoloma frowardii, efficiently produces manganese peroxidase (MNP) and is used for degradation of natural, persistent aromatic polymers (lignin, humic acids and brown coal components). The N. frowardii MNP has shown good activity in conversion of xenobiotic compounds such as polycyclic hydrocarbons and trinitrotoluene. However, this biotechnologically promising fungus has not previously been studied at the molecular biology level. We show here that according to the molecular characterization of its main MNP isozyme, Nf b19 MNP2, and partial sequencing of its MNP3-, three lignin peroxidase- and two laccase-encoding genes, and the gene encoding the ribosomal SSU 18S RNA, that the fungus has a close phylogenetic relationship to the white-rot basidiomycete Phlebia radiata (Fr.). Ribosomal internal transcribed spacer (ITS) sequence (ITS1+5.8S+ITS2) phylogeny reclassifies Nf b19 as a possible representative of a new species of the genus Phlebia, nearest to the Phlebia acerina clade. The genus Phlebia belongs to a completely different family (Corticiaceae) and order (Aphyllophorales) within the phylum Basidiomycota than the genus Nematoloma, which is classified in the order Agaricales, family Strophariaceae. Our results thus indicate a need for systematic re-identification of the previously named N. frowardii isolate b19.

  5. Sequence-specific label-free nucleic acid biosensor for the detection of the hepatitis C virus genotype 1a using a disposable pencil graphite electrode.

    PubMed

    Donmez, Soner; Arslan, Fatma; Arslan, Halit

    2016-05-01

    In this paper, we demonstrate a simple, sensitive, inexpensive, disposable and label-free electrochemical nucleic acid biosensor for the detection of the hepatitis C virus genotype 1a (HCV1a). The nucleic acid biosensor was designed with the amino-linked inosine-substituted 20-mer probes, which were immobilized onto a disposable pencil graphite electrode (PGE) by covalent linking. The proposed nucleic acid biosensor was linear in the range of 0.05 and 0.75 μM, exhibiting a limit of detection of 54.9 nM. The single-stranded synthetic PCR product analogs of HCV1a were also detected with satisfactory results under optimal conditions, showing the potential application of this biosensor. PMID:25619757

  6. Oncolytic Adenoviral Mutants with E1B19K Gene Deletions Enhance Gemcitabine-induced Apoptosis in Pancreatic Carcinoma Cells and Anti-Tumor Efficacy In vivo

    PubMed Central

    Leitner, Stephan; Sweeney, Katrina; Öberg, Daniel; Davies, Derek; Miranda, Enrique; Lemoine, Nick R.; Halldén, Gunnel

    2010-01-01

    Purpose Pancreatic adenocarcinoma is a rapidly progressive malignancy that is highly resistant to current chemotherapeutic modalities and almost uniformly fatal.We show that a novel targeting strategy combining oncolytic adenoviral mutants with the standard cytotoxic treatment, gemcitabine, can markedly improve the anticancer potency. Experimental Design Adenoviral mutants with the E1B19K gene deleted with and without E3B gene expression (AdΔE1B19K and dl337 mutants, respectively) were assessed for synergistic interactions in combination with gemcitabine. Cell viability, mechanism of cell death, and antitumor efficacy in vivo were determined in the pancreatic carcinoma cells PT45 and Suit2, normal human bronchial epithelial cells, and in PT45 xenografts. Results The ΔE1B19K-deleted mutants synergized with gemcitabine to selectively kill cultured pancreatic cancer cells and xenografts in vivo with no effect in normal cells. The corresponding wild-type virus (Ad5) stimulated drug-induced cell killing to a lesser degree. Gemcitabine blocked replication of all viruses despite the enhanced cell killing activity due to gemcitabine-induced delay in G1/S-cell cycle progression, with repression of cyclin E and cdc25A, which was not abrogated by viral E1A-expression. Synergistic cell death occurred through enhancement of gemcitabine-induced apoptosis in the presence of both AdΔE1B19K and dl337 mutants, shown by increased cell membrane fragmentation, caspase-3 activation, and mitochondrial dysfunction. Conclusions Our data suggest that oncolytic mutants lacking the antiapoptotic E1B19K gene can improve efficacy of DNA-damaging drugs such as gemcitabine through convergence on cellular apoptosis pathways.These findings imply that less toxic doses than currently practicedin the clinic could efficiently target pancreatic adenocarcinomas when combined with adenoviral mutants. PMID:19223497

  7. Clinical and epidemiological aspects of human parvovirus B19 infection in an urban area in Brazil (Niterói city area, State of Rio de Janeiro, Brazil).

    PubMed

    Oliveira, Solange Artimos de; Camacho, Luiz Antonio Bastos; Pereira, Antonio Carlos de Medeiros; Faillace, Tereza Filomena; Setubal, Sérgio; Nascimento, Jussara Pereira do

    2002-10-01

    This study was designed to analyse the clinical and epidemiological data from human parvovirus B19 cases in a six-year study of rash diseases conduct in an urban area in Brazil (Niterói city area, State of Rio de Janeiro). A total of 673 patients with acute rash diseases were seen at two primary health care units and at a general hospital. A clotted blood sample was collected from all subjects at the time of consultation. Forty-nine per cent (330 cases) of the patients were negative for dengue, rubella and measles IgM or for low avidity IgG to HHV-6. Of these 330, 105 (31.8%) were identified as IgM positive to parvovirus B19 by using an antibody capture EIA. During the study period, three distinct peaks of parvovirus infection were detected, suggesting that the disease appears to cycle in approximately 4-5 years. B19 infection was characterized by variable combinations of fever, flu-like symptoms, arthropathy, and gastrointestinal symptoms. Frequency of fever and arthropathy was substantially higher in adults, 75% [chi2 (1 D.F.) = 11.39, p = 0.0007] and 62.5% [chi2 (1 D.F.) = 29.89, p = 0.0000], respectively. "Slapped-cheek" appearance and reticular or lace-like rash were seen in only 30.1% of the children. No adult presented this typical rash. The lack of the typical rash pattern in a large proportion of parvovirus B19 and the similarity of clinical manifestations to other rash diseases, specially to rubella, highlight the difficulty of diagnosing B19 infection on clinical grounds alone.

  8. Comparison of a multiplexed MassARRAY system with real-time allele-specific PCR technology for genotyping of methicillin-resistant Staphylococcus aureus.

    PubMed

    Syrmis, M W; Moser, R J; Whiley, D M; Vaska, V; Coombs, G W; Nissen, M D; Sloots, T P; Nimmo, G R

    2011-12-01

    The Sequenom MassARRAY iPLEX single-nucleotide polymorphism (SNP) typing platform uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) coupled with single-base extension PCR for high-throughput multiplex SNP detection. In this study, we investigated the use of iPLEX MassARRAY technology for methicillin-resistant Staphylococcus aureus (MRSA) genotyping. A 16-plex MassARRAY iPLEX GOLD assay (MRSA-iPLEX) was developed that targets a set of informative SNPs and binary genes for MRSA characterization. The method was evaluated with 147 MRSA isolates, and the results were compared with those of an established SYBR Green-based real-time PCR system utilizing the same SNP-binary markers. A total of 2352 markers belonging to 44 SNP-binary profiles were analysed by both real-time PCR and MRSA-iPLEX. With real-time PCR as the reference standard, MRSA-iPLEX correctly assigned 2298 of the 2352 (97.7%) markers. Sequence variation in the MRSA-iPLEX primer targets accounted for the majority of MRSA-iPLEX erroneous results, highlighting the importance of primer target selection. MRSA-iPLEX provided optimal throughput for MRSA genotyping, and was, on a reagent basis, more cost-effective than the real-time PCR methods. The 16-plex MRSA-iPLEX is a suitable alternative to SYBR Green-based real-time PCR typing of major sequence types and clonal complexes of MRSA.

  9. Response of a Habitat-Forming Marine Plant to a Simulated Warming Event Is Delayed, Genotype Specific, and Varies with Phenology

    PubMed Central

    Reynolds, Laura K.; DuBois, Katherine; Abbott, Jessica M.; Williams, Susan L.; Stachowicz, John J.

    2016-01-01

    Growing evidence shows that increasing global temperature causes population declines and latitudinal shifts in geographical distribution for plants living near their thermal limits. Yet, even populations living well within established thermal limits of a species may suffer as the frequency and intensity of warming events increase with climate change. Adaptive response to this stress at the population level depends on the presence of genetic variation in thermal tolerance in the populations in question, yet few data exist to evaluate this. In this study, we examined the immediate effects of a moderate warming event of 4.5°C lasting 5 weeks and the legacy effects after a 5 week recovery on different genotypes of the marine plant Zostera marina (eelgrass). We conducted the experiment in Bodega Bay, CA USA, where average summer water temperatures are 14–15°C, but extended warming periods of 17–18°C occur episodically. Experimental warming increased shoot production by 14% compared to controls held at ambient temperature. However, after returning temperature to ambient levels, we found strongly negative, delayed effects of warming on production: shoot production declined by 27% and total biomass decreased by 50% relative to individuals that had not been warmed. While all genotypes’ production decreased in the recovery phase, genotypes that grew the most rapidly under benign thermal conditions (control) were the most susceptible to the detrimental effects of warming. This suggests a potential tradeoff in relative performance at normal vs. elevated temperatures. Modest short-term increases in water temperature have potentially prolonged negative effects within the species’ thermal envelope, but genetic variation within these populations may allow for population persistence and adaptation. Further, intraspecific variation in phenology can result in maintenance of population diversity and lead to enhanced production in diverse stands given sufficient frequency of

  10. Ligation-rolling circle amplification combined with γ-cyclodextrin mediated stemless molecular beacon for sensitive and specific genotyping of single-nucleotide polymorphism.

    PubMed

    Zou, Zhen; Qing, Zhihe; He, Xiaoxiao; Wang, Kemin; He, Dinggeng; Shi, Hui; Yang, Xue; Qing, Taiping; Yang, Xiaoxiao

    2014-07-01

    A novel approach for highly sensitive and selective genotyping of single-nucleotide polymorphism (SNP) has been developed based on ligation-rolling circle amplification (L-RCA) and stemless molecular beacon. In this approach, two tailored DNA probes were involved. The stemless molecular beacon, formed through the inclusion interactions of γ-cyclodextrin (γ-CD) and bis-pyrene labeled DNA fragment, was served as signal probe. In the absence of mutant target, the two pyrene molecules were bound in the γ-CD cavity to form an excimer and showed a strong fluorescence at 475 nm. It was here named γ-CD-P-MB. The padlock DNA probe was designed as recognition probe. Upon the recognition of a point mutation DNA targets, the padlock probe was ligated to generate a circular template. An RCA amplification was then initiated using the circular template in the presence of Phi29 polymerase and dNTPs. The L-RCA products, containing repetitive sequence units, subsequently hybridized with the γ-CD-P-MB. This made pyrene molecules away from γ-CD cavity and caused a decrease of excimer fluorescence. As a proof-of-concept, SNP typing of β-thalassemia gene at position -28 was investigated using this approach. The detection limit of mutated target was determined to be 40 fM. In addition, DNA ligase offered high fidelity in distinguishing the mismatched bases at the ligation site, resulting in positive detection of mutant target even when the ratio of the wildtype to the mutant is 999:1. Given these attractive characteristics, the developed approach might provide a great genotyping platform for pathogenic diagnosis and genetic analysis.

  11. Design and Assessment of a Real Time Reverse Transcription-PCR Method to Genotype Single-Stranded RNA Male-Specific Coliphages (Family Leviviridae).

    EPA Science Inventory

    A real-time, reverse transcription-PCR (RT-qPCR) assay was developed to differentiate the four genogroups of male-specific ssRNA coliphages (FRNA) (family Leviviridae). As FRNA display a trend of source-specificity (human sewage or animal waste) at the genogroup level, this assa...

  12. Self-accommodation of B19' martensite in Ti-Ni shape memory alloys - Part I. Morphological and crystallographic studies of the variant selection rule

    NASA Astrophysics Data System (ADS)

    Nishida, M.; Nishiura, T.; Kawano, H.; Inamura, T.

    2012-06-01

    The self-accommodation morphologies of B19‧ martensite in Ti-Ni alloys have been investigated by optical microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Twelve pairs of minimum units consisting of two habit plane variants (HPVs) with V-shaped morphology connected to a ? B19‧ type I variant accommodation twin were observed. Three types of self-accommodation morphologies, based on the V-shaped minimum unit, developed around one of the {111}B2 traces, which were triangular, rhombic and hexangular and consisted of three, four and six HPVs, respectively. In addition, the variant selection rule and the number of possible HPV combinations in each of these self-accommodation morphologies are discussed.

  13. The Determinants for the Enzyme Activity of Human Parvovirus B19 Phospholipase A2 (PLA2) and Its Influence on Cultured Cells

    PubMed Central

    Yi, Qianhui; Huang, Yu; Zhao, Dan; Yang, Yongbo; Tijssen, Peter; Qiu, Jianming; Liu, Kaiyu; Li, Yi

    2013-01-01

    Human parvovirus B19 (B19V) is the causative agent of erythema infectiosum in humans. B19 infection also causes severe disease manifestations, such as chronic anemia in immunocompromised patients, aplastic crisis in patients with a high turnover rate of red blood cells, and hydrops fetalis in pregnant women. Although a secreted phospholipase A2 (PLA2) motif has been identified in the unique region of the B19V minor capsid protein VP1(VP1u), the determinants for its enzyme activity and its influences on host cells are not well understood. The purpose of this study was to investigate the contribution of the PLA2 motif and other regions of the VP1u to the PLA2 activity, to determine the cellular localization of the VP1u protein, and to examine the effects of VP1u on cellular cytokines. We found that in addition to the critical conserved and non-conserved amino acids within the VP1u PLA2 motif, amino acid residues outside the VP1u PLA2 motif are also important for the PLA2 activity. VP1u and various mutants all revealed a nucleo-cytoplasmic distribution. UT7-Epo cells treated with prokaryotic expressed VP1u or mutant proteins with PLA2 activity released a large amount of free fatty acid (FFA), and the cell morphological change occurred dramatically. However, neither free fatty acid nor cell morphology change occurred for cells treated with the mutants without PLA2 activity. The wild type and the VP1u mutants with the PLA2 activity also activated TNF-α promoter and upregulated the transcription activity of NF-κB in transfected cells. In addition, we found that the amino acids outside the PLA2 domain are critical for the viral PLA2 activity, and that these tested VP1u mutants did not affect the localization of the VP1u protein. PMID:23596524

  14. The determinants for the enzyme activity of human parvovirus B19 phospholipase A2 (PLA2) and its influence on cultured cells.

    PubMed

    Deng, Xuefeng; Dong, Yanming; Yi, Qianhui; Huang, Yu; Zhao, Dan; Yang, Yongbo; Tijssen, Peter; Qiu, Jianming; Liu, Kaiyu; Li, Yi

    2013-01-01

    Human parvovirus B19 (B19V) is the causative agent of erythema infectiosum in humans. B19 infection also causes severe disease manifestations, such as chronic anemia in immunocompromised patients, aplastic crisis in patients with a high turnover rate of red blood cells, and hydrops fetalis in pregnant women. Although a secreted phospholipase A2 (PLA2) motif has been identified in the unique region of the B19V minor capsid protein VP1(VP1u), the determinants for its enzyme activity and its influences on host cells are not well understood. The purpose of this study was to investigate the contribution of the PLA2 motif and other regions of the VP1u to the PLA2 activity, to determine the cellular localization of the VP1u protein, and to examine the effects of VP1u on cellular cytokines. We found that in addition to the critical conserved and non-conserved amino acids within the VP1u PLA2 motif, amino acid residues outside the VP1u PLA2 motif are also important for the PLA2 activity. VP1u and various mutants all revealed a nucleo-cytoplasmic distribution. UT7-Epo cells treated with prokaryotic expressed VP1u or mutant proteins with PLA2 activity released a large amount of free fatty acid (FFA), and the cell morphological change occurred dramatically. However, neither free fatty acid nor cell morphology change occurred for cells treated with the mutants without PLA2 activity. The wild type and the VP1u mutants with the PLA2 activity also activated TNF-α promoter and upregulated the transcription activity of NF-κB in transfected cells. In addition, we found that the amino acids outside the PLA2 domain are critical for the viral PLA2 activity, and that these tested VP1u mutants did not affect the localization of the VP1u protein.

  15. Gene expression analysis of potential genes and pathways involved in the pathogenic mechanisms of parvovirus B19 in human colorectal cancer

    PubMed Central

    ZHANG, WEI-PING; YANG, HUA; CHEN, HONG; ZHU, HAI-RONG; LEI, QUAN; SONG, YUN-HONG; DAI, ZHONG-MING; SUN, JING-SHAN; JIANG, LI-LI; NIE, ZHAN-GUO

    2014-01-01

    In order to investigate the pathogenic mechanisms of parvovirus B19 in human colorectal cancer, plasmids containing the VP1 or VP2 viral capsid proteins or the NS1 non-structural proteins of parvovirus B19 were constructed and transfected into primary human colorectal epithelial cells and LoVo cells. Differential gene expression was detected using a human genome expression array. Functional gene annotation analyses were performed using Database for Annotation, Visualization and Integrated Discovery v6.7 software. Gene ontology (GO) analyses revealed that VP1-related functions included the immune response, immune system process, defense response and the response to stimulus, while NS1-associated functions were found to include organelle fission, nuclear division, mitosis, the M-phase of the mitotic cell cycle, the mitotic cell cycle, M-phase, cell cycle phase, cell cycle process and cell division. Pathway expression analysis revealed that VP1-associated pathways included cell adhesion molecules, antigen processing and presentation, cytokines and the inflammatory response. Moreover, NS1-associated pathways included the cell cycle, pathways in cancer, colorectal cancer, the wnt signaling pathway and focal adhesion. Among the differential genes detected in the present study, 12 genes were found to participate in general cancer pathways and six genes were observed to participate in colorectal cancer pathways. NS1 is a key molecule in the pathogenic mechanism of parvovirus B19 in colorectal cancer. Several GO categories, pathways and genes were selected and may be the key targets through which parvovirus B19 participates in colorectal cancer pathogenesis. PMID:25013465

  16. Peptide display on a surface loop of human parvovirus B19 VP2: Assembly and characterization of virus-like particles.

    PubMed

    Santillán-Uribe, José Sebastián; Valadez-García, Josefina; Morán-García, Areli Del Carmen; Santillán-Uribe, Hugo César; Bustos-Jaimes, Ismael

    2015-04-01

    Virus-like particles (VLPs) are valuable tools for nanotechnology and nanomedicine. These particles are obtained by the self-assembly, either in vivo or in vitro, of structural proteins of viral capsids. VLPs are excellent scaffolds for surface display of molecules. The N-termini of the structural proteins of human parvovirus B19 (B19V) have been already modified to display peptides or proteins. However, other surface-exposed elements have not been studied as potential locations for peptide display. In this research, we tested the potential of surface loop 62-75 of VP2 protein for the presentation of a 64-residue heterologous peptide. The chimeric protein was able to self-assemble in vitro into VLPs. Improved colloidal stability was observed for these particles, indicating that the peptide is on the surface of the particle. AFM analysis of the chimeric particles shows no obvious difference between the surfaces of particles assembled with VP2 and those assembled with the chimeric VP2. Our results indicate that loop 62-75 is a good candidate for heterologous peptide presentation on the surface of B19V VLPs. PMID:25701743

  17. Hepatitis C Genotype Influences Post-Liver Transplant Outcomes

    PubMed Central

    Campos-Varela, Isabel; Lai, Jennifer C.; Verna, Elizabeth C.; O'Leary, Jacqueline G.; Stravitz, R. Todd; Forman, Lisa M.; Trotter, James F.; Brown, Robert S.; Terrault, Norah A.

    2015-01-01

    Background In non-transplant patients with chronic hepatitis C virus (HCV), HCV genotype has been linked with a differential response to antiviral therapy, risk of steatosis and fibrosis, as well as all-cause mortality, but the role of HCV genotypes in post-transplant disease progression is less clear. Methods Using the multicenter CRUSH-C cohort, genotype-specific rates of advanced fibrosis, HCV-specific graft loss and, response of antiviral therapy were examined. Results Among 745 recipients [605 (81%) genotype 1, 53 (7%) genotype 2, and 87 (12%) genotype 3] followed for a median of 3.1 years (range 2.0-8.0) the unadjusted cumulative rate of advanced fibrosis at 3 years was 31%, 19% and 19% for genotypes 1, 2 and 3 (p=0.008). After multivariable adjustment, genotype remained a significant predictor, with genotype 2 having a 66% lower risk (p=0.02) and genotype 3 having a 41% lower risk (p=0.07) of advanced fibrosis compared to genotype 1 patients. The cumulative 5-year rates of HCV-specific graft survival were 84%, 90% and 94% for genotypes 1, 2 and 3, p=0.10. A total of 37% received antiviral therapy, with higher rates of sustained virologic response in patients with genotype 2 (HR=5.10; p=0.003) and genotype 3 (HR=3.27; p=0.006) compared to patients with genotype 1. Conclusion Risk of advanced fibrosis and response to therapy are strongly influenced by genotype. LT recipients with HCV genotype 1 have the highest risk of advanced fibrosis and lowest SVR rate. These findings highlight the need for genotype-specific management strategies. PMID:25211520

  18. Cloning to reproduce desired genotypes.

    PubMed

    Westhusin, M E; Long, C R; Shin, T; Hill, J R; Looney, C R; Pryor, J H; Piedrahita, J A

    2001-01-01

    Cloned sheep, cattle, goats, pigs and mice have now been produced using somatic cells for nuclear transplantation. Animal cloning is still very inefficient with on average less than 10% of the cloned embryos transferred resulting in a live offspring. However successful cloning of a variety of different species and by a number of different laboratory groups has generated tremendous interest in reproducing desired genotypes. Some of these specific genotypes represent animal cell lines that have been genetically modified. In other cases there is a significant demand for cloning animals characterized by their inherent genetic value, for example prize livestock, household pets and rare or endangered species. A number of different variables may influence the ability to reproduce a specific genotype by cloning. These include species, source of recipient ova, cell type of nuclei donor, treatment of donor cells prior to nuclear transfer, and the techniques employed for nuclear transfer. At present, there is no solid evidence that suggests cloning will be limited to only a few specific animals, and in fact, most data collected to date suggests cloning will be applicable to a wide variety of different animals. The ability to reproduce any desired genotype by cloning will ultimately depend on the amount of time and resources invested in research.

  19. Hepatitis C virus genotypes in Myanmar

    PubMed Central

    Win, Nan Nwe; Kanda, Tatsuo; Nakamoto, Shingo; Yokosuka, Osamu; Shirasawa, Hiroshi

    2016-01-01

    Myanmar is adjacent to India, Bangladesh, Thailand, Laos and China. In Myanmar, the prevalence of hepatitis C virus (HCV) infection is 2%, and HCV infection accounts for 25% of hepatocellular carcinoma. In this study, we reviewed the prevalence of HCV genotypes in Myanmar. HCV genotypes 1, 3 and 6 were observed in volunteer blood donors in and around the Myanmar city of Yangon. Although there are several reports of HCV genotype 6 and its variants in Myanmar, the distribution of the HCV genotypes has not been well documented in areas other than Yangon. Previous studies showed that treatment with peginterferon and a weight-based dose of ribavirin for 24 or 48 wk could lead to an 80%-100% sustained virological response (SVR) rates in Myanmar. Current interferon-free treatments could lead to higher SVR rates (90%-95%) in patients infected with almost all HCV genotypes other than HCV genotype 3. In an era of heavy reliance on direct-acting antivirals against HCV, there is an increasing need to measure HCV genotypes, and this need will also increase specifically in Myanmar. Current available information of HCV genotypes were mostly from Yangon and other countries than Myanmar. The prevalence of HCV genotypes in Myanmar should be determined. PMID:27468202

  20. The dual effect of adenovirus type 5 E1A 13S protein on NF-kappaB activation is antagonized by E1B 19K.

    PubMed Central

    Schmitz, M L; Indorf, A; Limbourg, F P; Städtler, H; Traenckner, E B; Baeuerle, P A

    1996-01-01

    The genomes of human adenoviruses encode several regulatory proteins, including the two differentially spliced gene products E1A and E1B. Here, we show that the 13S but not the 12S splice variant of E1A of adenovirus type 5 can activate the human transcription factor NF-kappaB in a bimodal fashion. One mode is the activation of NF-kappaB containing the p65 subunit from the cytoplasmic NF-kappaB-IkappaB complex. This activation required reactive oxygen intermediates and the phosphorylation of IkappaBalpha at serines 32 and 36, followed by IkappaBalpha degradation and the nuclear uptake of NF-kappaB. In addition, 13S E1A stimulated the transcriptional activity of the C-terminal 80 amino acids of p65 at a core promoter with either a TATA box or an initiator (INR) element. The C-terminal 80 amino acids of p65 were found to associate with E1A in vitro. The activation of NF-kappaB-dependent reporter gene transcription by E1A was potently suppressed upon coexpression of the E1B 19-kDa protein (19K). E1B 19K prevented both the activation of NF-kappaB and the E1A-mediated transcriptional enhancement of p65. These inhibitory effects were not found for the 55-kDa splice variant of the E1B protein. We suggest that the inductive effect of E1A 13S on the host factor NF-kappaB, whose activation is important for the transcription of various adenovirus genes, must be counteracted by the suppressive effect of E1B 19K so that the adenovirus-infected cell can escape the immune-stimulatory and apoptotic effects of NF-kappaB. PMID:8754803

  1. Priming-mediated systemic resistance in cucumber induced by Pseudomonas azotoformans GC-B19 and Paenibacillus elgii MM-B22 against Colletotrichum orbiculare.

    PubMed

    Sang, Mee Kyung; Kim, Eui Nam; Han, Gyung Deok; Kwack, Min Sun; Jeun, Yong Chull; Kim, Ki Deok

    2014-08-01

    Induced systemic resistance (ISR) can be activated by biotic agents, including root-associated beneficial bacteria to inhibit pathogen infection. We investigated priming-mediated ISR in cucumber induced by Pseudomonas azotoformans GC-B19 and Paenibacillus elgii MM-B22 against Colletotrichum orbiculare (causal fungus of anthracnose). In addition, we examined whether this ISR expression was bacterial density-dependent by assessing peroxidase activity in the presence and absence of the pathogen. As a result, root treatment with the ISR-eliciting strains GC-B19 and MM-B22 or the chemical inducer DL-β-amino-n-butyric acid (positive control) significantly inhibited fungal infection process (conidial germination and appressorium formation) and disease severity compared with the non-ISR-eliciting strain, Pseudomonas aeruginosa PK-B09 (negative control), and MgSO4 solution (untreated control). These treatments effectively induced rapid elicitation of hypersensitive reaction-like cell death with H2O2 generations, and accumulation of defense-related enzymes (β-1,3-glucanase, chitinase, and peroxidase) in cucumber leaves in the "primed" state against C. orbiculare. In addition, ISR expression was dependent on the bacterial cell density in the rhizosphere. This ISR expression was derived from the presence of sustained bacterial populations ranging from 10(4) to 10(6) cells/g of potting mix over a period of time after introduction of bacteria (10(6) to 10(10) cells/g of potting mix) into the rhizosphere. Taken together, these results suggest that priming-mediated ISR against C. orbiculare in cucumber can be induced in a bacterial density-dependent manner by Pseudomonas azotoformans GC-B19 and Paenibacillus elgii MM-B22. PMID:24502209

  2. A simple and rapid method for HLA-DQA1 genotyping by digestion of PCR-amplified DNA with allele specific restriction endonucleases.

    PubMed

    Maeda, M; Murayama, N; Ishii, H; Uryu, N; Ota, M; Tsuji, K; Inoko, H

    1989-11-01

    The second exon of the HLA-DQA1 genes was selectively amplified from genomic DNAs of 72 HLA-homozygous B cell lines by the polymerase chain reaction (PCR). Amplified DNAs were digested with HaeIII, Ddel, ScrFI, FokI and RsaI, which recognize allelic sequence variations in the polymorphic segments of the DQA1 second exon, and then subjected to electrophoresis in polyacrylamide gels. Eight different polymorphic patterns of restriction fragments were obtained, and seven were identical to patterns predicted from the known DNA sequences, correlating with each HLA-DQw type defined by serological typing. The remaining one pattern cannot be explained from the sequence data, suggesting the presence of a novel DQA1 allele at the nucleotide level. This PCR-RFLP method provides a simple and rapid technique for accurate definition of the HLA-DQ types at the nucleotide level, eliminating the need for radioisotope as well as allele specific oligonucleotide probes and can be extended and applied to HLA-DR, -Dw DP typing. PMID:2576477

  3. Large-Scale SNP Discovery and Genotyping for Constructing a High-Density Genetic Map of Tea Plant Using Specific-Locus Amplified Fragment Sequencing (SLAF-seq).

    PubMed

    Ma, Jian-Qiang; Huang, Long; Ma, Chun-Lei; Jin, Ji-Qiang; Li, Chun-Fang; Wang, Rong-Kai; Zheng, Hong-Kun; Yao, Ming-Zhe; Chen, Liang

    2015-01-01

    Genetic maps are important tools in plant genomics and breeding. The present study reports the large-scale discovery of single nucleotide polymorphisms (SNPs) for genetic map construction in tea plant. We developed a total of 6,042 valid SNP markers using specific-locus amplified fragment sequencing (SLAF-seq), and subsequently mapped them into the previous framework map. The final map contained 6,448 molecular markers, distributing on fifteen linkage groups corresponding to the number of tea plant chromosomes. The total map length was 3,965 cM, with an average inter-locus distance of 1.0 cM. This map is the first SNP-based reference map of tea plant, as well as the most saturated one developed to date. The SNP markers and map resources generated in this study provide a wealth of genetic information that can serve as a foundation for downstream genetic analyses, such as the fine mapping of quantitative trait loci (QTL), map-based cloning, marker-assisted selection, and anchoring of scaffolds to facilitate the process of whole genome sequencing projects for tea plant.

  4. Species specificity and interspecies relatedness in VP4 genotypes demonstrated by VP4 sequence analysis of equine, feline, and canine rotavirus strains.

    PubMed

    Taniguchi, K; Urasawa, T; Urasawa, S

    1994-05-01

    We determined the nucleotide and deduced amino acid sequences of the VP4 genes of five equine, two feline, and two canine rotavirus strains. A high degree of homology (> 97.0%) was found among the VP4 amino acid sequences of the equine strains H2, FI-14, and FI23. Equine strain L338 has a distinct VP4 amino acid sequence from those of the other equine strains (78.1% or less homology), and the L338 VP4 exhibited more than 17.0% divergence at the amino acid level from those of rotavirus strains published so far. The VP4 amino acid sequence of equine strain H1, which showed low homology with those of other equine strains, shares > 95.4% homology to those of porcine strains OSU and YM. VP4 amino acid sequences of feline strain Cat97 and canine strains CU-1 and K9 showed a high degree of homology (96.8 to 97.2%) to one another, and were found to be quite similar (96.0-97.0% homology) to that of a human HCR3 strain recently characterized. Feline strain Cat2, whose VP4 sequence is distinct from that of strain Cat97, has a VP4 similar to those of human strains K8 and AU-1 (97.8 and 97.5% homologies at amino acid level, respectively). Thus, the VP4 sequences of rotaviruses showed species specificity and interspecies relatedness. PMID:8178429

  5. Comparison of Two Widely Used Human Papillomavirus Detection and Genotyping Methods, GP5+/6+-Based PCR Followed by Reverse Line Blot Hybridization and Multiplex Type-Specific E7-Based PCR.

    PubMed

    Clifford, Gary M; Vaccarella, Salvatore; Franceschi, Silvia; Tenet, Vanessa; Umulisa, M Chantal; Tshomo, Ugyen; Dondog, Bolormaa; Vorsters, Alex; Tommasino, Massimo; Heideman, Daniëlle A M; Snijders, Peter J F; Gheit, Tarik

    2016-08-01

    GP5+/6+-based PCR followed by reverse line blot hybridization (GP5+/6+RLB) and multiplex type-specific PCR (E7-MPG) are two human papillomavirus (HPV) genotyping methodologies widely applied in epidemiological research. We investigated their relative analytical performance in 4,662 samples derived from five studies in Bhutan, Rwanda, and Mongolia coordinated by the International Agency for Research on Cancer (IARC). A total of 630 samples were positive by E7-MPG only (13.5%), 24 were positive by GP5+/6+RLB only (0.5%), and 1,014 were positive (21.8%) by both methods. Ratios of HPV type-specific positivity of the two tests (E7-MPG:GP5+/6+RLB ratio) were calculated among 1,668 samples that were HPV positive by one or both tests. E7-MPG:GP5+/6+RLB ratios were >1 for all types and highly reproducible across populations and sample types. E7-MPG:GP5+/6+RLB ratios were highest for HPV53 (7.5) and HPV68 (7.1). HPV16 (1.6) and HPV18 (1.7) had lower than average E7-MPG:GP5+/6+RLB ratios. Among E7-MPG positive infections, median mean fluorescence intensity (MFI; a semiquantitative measure of viral load) tended to be higher among samples positive for the same virus type by GP5+/6+RLB than for those negative for the same type by GP5+/6+RLB. Exceptions, however, included HPV53, -59, and -82, for which the chances of being undetected by GP5+/6+RLB appeared to be MFI independent. Furthermore, the probability of detecting an additional type by E7-MPG was higher when another type was already detected by GP5+/6+RLB, suggesting the existence of masking effects due to competition for GP5+/6+ PCR primers. In conclusion, this analysis is not an evaluation of clinical performance but may inform choices for HPV genotyping methods in epidemiological studies, when the relative merits and dangers of sensitivity versus specificity for individual types should be considered, as well as the potential to unmask nonvaccine types following HPV vaccination. PMID:27225411

  6. Self-accommodation of B19' martensite in Ti-Ni shape memory alloys. Part III. Analysis of habit plane variant clusters by the geometrically nonlinear theory

    NASA Astrophysics Data System (ADS)

    Inamura, T.; Nishiura, T.; Kawano, H.; Hosoda, H.; Nishida, M.

    2012-06-01

    Competition between the invariant plane (IP) condition at the habit plane, the twin orientation relation (OR) and the kinematic compatibility (KC) at the junction plane (JP) of self-accommodated B19‧ martensite in Ti-Ni was investigated via the geometrically nonlinear theory to understand the habit plane variant (HPV) clusters presented in Parts I and II of this work. As the IP condition cannot be satisfied simultaneously with KC, an additional rotation Q is necessary to form compatible JPs for all HPV pairs. The rotation J necessary to form the exact twin OR between the major correspondence variants (CVs) in each HPV was also examined. The observed HPV cluster was not the cluster with the smallest Q but the one satisfying Q = J with a { ? 1}B19‧ type I twin at JP. Both Q and J are crucial to understanding the various HPV clusters in realistic transformations. Finally, a scheme for the ideal HPV cluster composed of six HPVs is also proposed.

  7. HCV genotyping from NGS short reads and its application in genotype detection from HCV mixed infected plasma.

    PubMed

    Qiu, Ping; Stevens, Richard; Wei, Bo; Lahser, Fred; Howe, Anita Y M; Klappenbach, Joel A; Marton, Matthew J

    2015-01-01

    Genotyping of hepatitis C virus (HCV) plays an important role in the treatment of HCV. As new genotype-specific treatment options become available, it has become increasingly important to have accurate HCV genotype and subtype information to ensure that the most appropriate treatment regimen is selected. Most current genotyping methods are unable to detect mixed genotypes from two or more HCV infections. Next generation sequencing (NGS) allows for rapid and low cost mass sequencing of viral genomes and provides an opportunity to probe the viral population from a single host. In this paper, the possibility of using short NGS reads for direct HCV genotyping without genome assembly was evaluated. We surveyed the publicly-available genetic content of three HCV drug target regions (NS3, NS5A, NS5B) in terms of whether these genes contained genotype-specific regions that could predict genotype. Six genotypes and 38 subtypes were included in this study. An automated phylogenetic analysis based HCV genotyping method was implemented and used to assess different HCV target gene regions. Candidate regions of 250-bp each were found for all three genes that have enough genetic information to predict HCV genotypes/subtypes. Validation using public datasets shows 100% genotyping accuracy. To test whether these 250-bp regions were sufficient to identify mixed genotypes, we developed a random primer-based method to sequence HCV plasma samples containing mixtures of two HCV genotypes in different ratios. We were able to determine the genotypes without ambiguity and to quantify the ratio of the abundances of the mixed genotypes in the samples. These data provide a proof-of-concept that this random primed, NGS-based short-read genotyping approach does not need prior information about the viral population and is capable of detecting mixed viral infection.

  8. Analysis of nucleotide sequences of human parvovirus B19 genome reveals two different modes of evolution, a gradual alteration and a sudden replacement: a retrospective study in Sapporo, Japan, from 1980 to 2008.

    PubMed

    Suzuki, Masashi; Yoto, Yuko; Ishikawa, Aki; Tsutsumi, Hiroyuki

    2009-11-01

    There have been no long-term systematic analyses of the molecular epidemiology of human parvovirus B19 (B19V). We investigated the variations of nucleotide sequences of B19V strains collected in Sapporo, Japan, from 1980 to 2008. In that period, six outbreaks of erythema infectiosum occurred regularly at 5-year intervals. The B19V strains collected successively, regardless of the outbreak, were analyzed for nucleotide variation in the subgenomic NS1-VP1u junction. The isolated strains can be classified into 10 subgroups. Two patterns of change of endemic strains were observed. One was a dynamic replacement of strains that occurred almost every 10 years, and the other was a gradual change consisting of an accumulation of point mutations.

  9. Analysis of Nucleotide Sequences of Human Parvovirus B19 Genome Reveals Two Different Modes of Evolution, a Gradual Alteration and a Sudden Replacement: a Retrospective Study in Sapporo, Japan, from 1980 to 2008▿

    PubMed Central

    Suzuki, Masashi; Yoto, Yuko; Ishikawa, Aki; Tsutsumi, Hiroyuki

    2009-01-01

    There have been no long-term systematic analyses of the molecular epidemiology of human parvovirus B19 (B19V). We investigated the variations of nucleotide sequences of B19V strains collected in Sapporo, Japan, from 1980 to 2008. In that period, six outbreaks of erythema infectiosum occurred regularly at 5-year intervals. The B19V strains collected successively, regardless of the outbreak, were analyzed for nucleotide variation in the subgenomic NS1-VP1u junction. The isolated strains can be classified into 10 subgroups. Two patterns of change of endemic strains were observed. One was a dynamic replacement of strains that occurred almost every 10 years, and the other was a gradual change consisting of an accumulation of point mutations. PMID:19710152

  10. Frequency of Natural Resistance within NS5a Replication Complex Domain in Hepatitis C Genotypes 1a, 1b: Possible Implication of Subtype-Specific Resistance Selection in Multiple Direct Acting Antivirals Drugs Combination Treatment.

    PubMed

    Bagaglio, Sabrina; Andolina, Andrea; Merli, Marco; Uberti-Foppa, Caterina; Morsica, Giulia

    2016-04-01

    Different HCV subtypes may naturally harbor different resistance selection to anti-NS5a inhibitors. 2761 sequences retrieved from the Los Alamos HCV database were analyzed in the NS5a domain 1, the target of NS5a inhibitors. The NS5a resistance-associated polymorphisms (RAPs) were more frequently detected in HCV G1b compared to G1a. The prevalence of polymorphisms associated with cross-resistance to compounds in clinical use (daclatasvir, DCV, ledipasvir, LDV, ombitasvir, and OMV) or scheduled to come into clinical use in the near future (IDX719, elbasvir, and ELV) was higher in G1b compared to G1a (37/1552 (2.4%) in 1b sequences and 15/1209 (1.2%) in 1a isolates, p = 0.040). Interestingly, on the basis of the genotype-specific resistance pattern, 95 (6.1%) G1b sequences had L31M RAP to DCV/IDX719, while 6 sequences of G1a (0.5%) harbored L31M RAP, conferring resistance to DCV/LDV/IDX719/ELV (p < 0.0001). Finally, 28 (2.3%) G1a and none of G1b isolates harbored M28V RAP to OMV (p < 0.0001). In conclusion, the pattern of subtype-specific resistance selection in the naturally occurring strains may guide the treatment option in association with direct acting antivirals (DAAs) targeting different regions, particularly in patients that are difficult to cure, such as those with advanced liver disease or individuals who have failed previous DAAs. PMID:27023593

  11. Distribution of Hepatitis C Virus Genotypes in Bahrain

    PubMed Central

    Janahi, Essam M.; Al-Mannai, Mariam; Singh, Hemlata; Jahromi, Mohamed M.

    2015-01-01

    Background: Approximately 170 million people are infected with Hepatitis C virus (HCV) worldwide, making it one of the world’s major infectious diseases. There are no published population based studies about the prevalence of HCV genotypes in Bahrain. Objectives: Therefore, the aim of the present study was to investigate the prevalence and distribution of HCV genotypes and subtypes among a large sample of patients with chronic HCV infection in Bahrain. Patients and Methods: Serum samples were collected from 202 HCV positive patients; of them 128 had a viral load (> 500 IU/mL) suitable for the type-specific genotyping assay. Gender-wise and age-wise differences in the distribution of HCV genotypes were determined by Chi Square and Fisher’s Exact tests. Results: The predominant genotype among Bahraini patients was type 1 (36.71%), followed by genotypes 3 and 4 (15.6% each) and the lowest frequency was found for genotype 2 (3.9%). Among genotype 1, subtype 1b had the highest frequency (21.09%), followed by subtype 1a (14.06%). Among genotype 3, subtype 3a had the highest frequency (11.72%), while among genotype 4, most of subtypes were undetermined. The frequency of all different HCV genotypes was higher in male patients compared to female patients. Genotype 1 was most common in the age group of 51 - 60 years (38.3%), genotype 2 in 21 - 30 years (60%) and genotype 3 in 51 - 60 years (30%), while genotype 4 was most frequent among the age group > 61 (40%). Conclusions: The most common HCV genotype in Bahrain was subtype 1b followed by 1a and 3a. Further studies involving sources of transmission in Bahrain are required to enhance control measures for HCV infection. PMID:26977163

  12. Global Distribution and Prevalence of Hepatitis C Virus Genotypes

    PubMed Central

    Messina, Jane P; Humphreys, Isla; Flaxman, Abraham; Brown, Anthony; Cooke, Graham S; Pybus, Oliver G; Barnes, Eleanor

    2015-01-01

    Hepatitis C virus (HCV) exhibits high genetic diversity, characterized by regional variations in genotype prevalence. This poses a challenge to the improved development of vaccines and pan-genotypic treatments, which require the consideration of global trends in HCV genotype prevalence. Here we provide the first comprehensive survey of these trends. To approximate national HCV genotype prevalence, studies published between 1989 and 2013 reporting HCV genotypes are reviewed and combined with overall HCV prevalence estimates from the Global Burden of Disease (GBD) project. We also generate regional and global genotype prevalence estimates, inferring data for countries lacking genotype information. We include 1,217 studies in our analysis, representing 117 countries and 90% of the global population. We calculate that HCV genotype 1 is the most prevalent worldwide, comprising 83.4 million cases (46.2% of all HCV cases), approximately one-third of which are in East Asia. Genotype 3 is the next most prevalent globally (54.3 million, 30.1%); genotypes 2, 4, and 6 are responsible for a total 22.8% of all cases; genotype 5 comprises the remaining <1%. While genotypes 1 and 3 dominate in most countries irrespective of economic status, the largest proportions of genotypes 4 and 5 are in lower-income countries. Conclusion: Although genotype 1 is most common worldwide, nongenotype 1 HCV cases—which are less well served by advances in vaccine and drug development—still comprise over half of all HCV cases. Relative genotype proportions are needed to inform healthcare models, which must be geographically tailored to specific countries or regions in order to improve access to new treatments. Genotype surveillance data are needed from many countries to improve estimates of unmet need. (Hepatology 2015;61:77–87) PMID:25069599

  13. Fifth Disease (Parvovirus B19)

    MedlinePlus

    ... Health Issues Conditions Abdominal ADHD Allergies & Asthma Autism Cancer Chest & Lungs Chronic Conditions Cleft & Craniofacial Developmental Disabilities Ear Nose & Throat Emotional Problems Eyes Fever From Insects or Animals Genitals and Urinary Tract Glands & Growth ...

  14. HIV Genotypic Resistance Testing

    MedlinePlus

    ... be limited. Home Visit Global Sites Search Help? HIV Antiretroviral Drug Resistance Testing, Genotypic Share this page: Was this page helpful? Also known as: Anti-retroviral Drug Resistance Testing; ARV Resistance Testing Formal name: ...

  15. APOE Genotyping, Cardiovascular Disease

    MedlinePlus

    ... Risk Assessment ; HDL Cholesterol ; LDL Cholesterol ; Lipid Profile ; Triglycerides Were you looking instead for APOE genotyping ordered ... the skin called xanthomas, a high level of triglycerides in the blood, and atherosclerosis that develops at ...

  16. Molecular and Functional Analyses of a Human Parvovirus B19 Infectious Clone Demonstrates Essential Roles for NS1, VP1, and the 11-Kilodalton Protein in Virus Replication and Infectivity

    PubMed Central

    Zhi, Ning; Mills, Ian P.; Lu, Jun; Wong, Susan; Filippone, Claudia; Brown, Kevin E.

    2006-01-01

    In an attempt to experimentally define the roles of viral proteins encoded by the B19 genome in the viral life cycle, we utilized the B19 infectious clone constructed in our previous study to create two groups of B19 mutant genomes: (i) null mutants, in which either a translational initiation codon for each of these viral genes was substituted by a translational termination codon or a termination codon was inserted into the open reading frame by a frameshift; and (ii) a deletion mutant, in which half of the hairpin sequence was deleted at both the 5′ and the 3′ termini. The impact of these mutations on viral infectivity, DNA replication, capsid protein production, and distribution was systematically examined. Null mutants of the NS and VP1 proteins or deletion of the terminal hairpin sequence completely abolished the viral infectivity, whereas blocking expression of the 7.5-kDa protein or the putative protein X had no effect on infectivity in vitro. Blocking expression of the proline-rich 11-kDa protein significantly reduced B19 viral infectivity, and protein studies suggested that the expression of the 11-kDa protein was critical for VP2 capsid production and trafficking in infected cells. These findings suggest a previously unrecognized role for the 11-kDa protein, and together the results enhance our understanding of the key features of the B19 viral genome and proteins. PMID:16731932

  17. A case of recurrent autoimmune hemolytic anemia during remission associated with acute pure red cell aplasia and hemophagocytic syndrome due to human parvovirus B19 infection successfully treated by steroid pulse therapy with a review of the literature.

    PubMed

    Sekiguchi, Yasunobu; Shimada, Asami; Imai, Hidenori; Wakabayashi, Mutsumi; Sugimoto, Keiji; Nakamura, Noriko; Sawada, Tomohiro; Komatsu, Norio; Noguchi, Masaaki

    2014-01-01

    The patient was a 47-year-old man diagnosed as having autoimmune hemolytic anemia (AIHA) in April 2011. He also had a congenital chromosomal abnormality, a balanced translocation. Treatment with prednisolone (PSL) 60 mg/day resulted in resolution of the AIHA, and the treatment was completed in November 2011. While the patient no longer had anemia, the direct and indirect Coombs tests remained positive. In May 2013, he developed recurrent AIHA associated with acute pure red cell aplasia (PRCA) and hemophagocytic syndrome (HPS) caused by human parvovirus B19 (HPV B19) infection. Tests for anti-erythropoietin and anti-erythropoietin receptor antibodies were positive. Steroid pulse therapy resulted in resolution of the AIHA, PRCA, as well as HPS. The serum test for anti-erythropoietin antibodies also became negative after the treatment. However, although the serum was positive for anti-HPV B19 IgG antibodies, the patient continued to have a low CD4 lymphocyte count (CD4, <300/μL) and persistent HPV B19 infection (HPV B19 DNA remained positive), suggesting the risk of recurrence and bone marrow failure.

  18. Circulation of HRSV in Belgium: from multiple genotype circulation to prolonged circulation of predominant genotypes.

    PubMed

    Houspie, Lieselot; Lemey, Philippe; Keyaerts, Els; Reijmen, Eva; Vergote, Valentijn; Vankeerberghen, Anne; Vaeyens, Freya; De Beenhouwer, Hans; Van Ranst, Marc

    2013-01-01

    Molecular surveillance of HRSV in Belgium for 15 consecutive seasons (1996-2011) revealed a shift from a regular 3-yearly cyclic pattern, into a yearly alternating periodicity where HRSV-B is replaced by HRSV-A. Phylogenetic analysis for HRSV-A demonstrated the stable circulation of GA2 and GA5, with GA2 being dominant over GA5 during 5 consecutive seasons (2006-2011). We also identified 2 new genotype specific amino acid mutations of the GA2 genotype (A122 and Q156) and 7 new GA5 genotype specific amino acid mutations (F102, I108, T111, I125, D161, S191 and L217). Several amino acid positions, all located in the second hypervariable region of HRSV-A were found to be under positive selection. Phylogenetic analysis of HRSV-B showed the circulation of GB12 and GB13, where GB13 represented 100% of the isolated strains in 4 out of 5 consecutive seasons (2007-2011). Amino acids under positive selection were all located in the aminoterminal hypervariable region of HRSV-B, except one amino acid located in the conserved region. The genotype distribution within the HRSV-B subgroup has evolved from a co-circulation of multiple genotypes to the circulation of a single predominant genotype. The Belgian GB13 strains circulating since 2006, all clustered under the BAIV branch and contained several branch specific amino acid substitutions. The demographic history of genotypes GA2, GA5 and GB13 demonstrated a decrease in the total GA2 and GA5 population size, coinciding with the global expansion of the GB13 population. The emergence of the GB13 genotype resulted in a newly established balance between the predominant genotypes. PMID:23577109

  19. Conformational Changes in the VP1-Unique Region of Native Human Parvovirus B19 Lead to Exposure of Internal Sequences That Play a Role in Virus Neutralization and Infectivity▿

    PubMed Central

    Ros, Carlos; Gerber, Marco; Kempf, Christoph

    2006-01-01

    The unique region of the capsid protein VP1 (VP1u) of human parvovirus B19 (B19) elicits a dominant immune response and has a phospholipase A2 (PLA2) activity, which is necessary for the infection. In contrast to the rest of the parvoviruses, the VP1u of B19 is thought to occupy an external position in the virion, making this region a promising candidate for vaccine development. By using a monoclonal antibody against the most-N-terminal portion of VP1u, we revealed that this region rich in neutralizing epitopes is not accessible in native capsids. However, exposure of capsids to increasing temperatures or low pH led to its progressive accessibility without particle disassembly. Although unable to bind free virus or to block virus attachment to the cell, the anti-VP1u antibody was neutralizing, suggesting that the exposure of the epitope and the subsequent virus neutralization occur only after receptor attachment. The measurement of the VP1u-associated PLA2 activity of B19 capsids revealed that this region is also internal but becomes exposed in heat- and in low-pH-treated particles. In sharp contrast to native virions, the VP1u of baculovirus-derived B19 capsids was readily accessible in the absence of any treatment. These results indicate that stretches of VP1u of native B19 capsids harboring neutralizing epitopes and essential functional motifs are not external to the capsid. However, a conformational change renders these regions accessible and triggers the PLA2 potential of the virus. The results also emphasize major differences in the VP1u conformation between natural and recombinant particles. PMID:17020940

  20. Conformational changes in the VP1-unique region of native human parvovirus B19 lead to exposure of internal sequences that play a role in virus neutralization and infectivity.

    PubMed

    Ros, Carlos; Gerber, Marco; Kempf, Christoph

    2006-12-01

    The unique region of the capsid protein VP1 (VP1u) of human parvovirus B19 (B19) elicits a dominant immune response and has a phospholipase A(2) (PLA(2)) activity, which is necessary for the infection. In contrast to the rest of the parvoviruses, the VP1u of B19 is thought to occupy an external position in the virion, making this region a promising candidate for vaccine development. By using a monoclonal antibody against the most-N-terminal portion of VP1u, we revealed that this region rich in neutralizing epitopes is not accessible in native capsids. However, exposure of capsids to increasing temperatures or low pH led to its progressive accessibility without particle disassembly. Although unable to bind free virus or to block virus attachment to the cell, the anti-VP1u antibody was neutralizing, suggesting that the exposure of the epitope and the subsequent virus neutralization occur only after receptor attachment. The measurement of the VP1u-associated PLA(2) activity of B19 capsids revealed that this region is also internal but becomes exposed in heat- and in low-pH-treated particles. In sharp contrast to native virions, the VP1u of baculovirus-derived B19 capsids was readily accessible in the absence of any treatment. These results indicate that stretches of VP1u of native B19 capsids harboring neutralizing epitopes and essential functional motifs are not external to the capsid. However, a conformational change renders these regions accessible and triggers the PLA(2) potential of the virus. The results also emphasize major differences in the VP1u conformation between natural and recombinant particles.

  1. Pure red cell aplasia due to parvovirus B19 infection after liver transplantation: A case report and review of the literature

    PubMed Central

    Liang, Ting-Bo; Li, Dong-Lin; Yu, Jun; Bai, Xue-Li; Liang, Liang; Xu, Shi-Guo; Wang, Wei-Lin; Shen, Yan; Zhang, Min; Zheng, Shu-Sen

    2007-01-01

    Pure red cell aplasia (PRCA) due to parvovirus B19 (PVB19) infection after solid organ transplantation has been rarely reported and most of the cases were renal transplant recipients. Few have been described after liver transplantation. Moreover, little information on the management of this easily recurring disease is available at present. We describe the first case of a Chinese liver transplant recipient with PVB19-induced PRCA during immunosuppressive therapy. The patient suffered from progressive anemia with the lowest hemoglobin level of 21 g/L. Bone marrow biopsy showed selectively inhibited erythropoiesis with giant pronormoblasts. Detection of PVB19-DNA in serum with quantitative polymerase chain reaction (PCR) revealed a high level of viral load. After 2 courses of intravenous immunoglobulin (IVIG) therapy, bone marrow erythropoiesis recovered with his hemoglobin level increased to 123 g/L. He had a low-level PVB19 load for a 5-mo follow-up period without recurrence of PRCA, and finally the virus was cleared. Our case indicates that clearance of PVB19 by IVIG in transplant recipients might be delayed after recovery of anemia. PMID:17461508

  2. Agranulocytosis under biotherapy in rheumatoid arthritis: three cases hypothesis of parvovirus B19 involvement in agranulocytosis observed under tocilizumab and rituximab for the treatment of rheumatoid arthritis.

    PubMed

    Giraud, C; Tatar, Z; Soubrier, M

    2016-10-01

    Leukopenia is a considerably common complication of tocilizumab [TCZ] and rituximab [RTX] therapy. RTX-induced leukopenia typically exhibits delayed onset. While agranulocytosis has been reported linked to RTX treatment of lymphoma, this complication rarely occurs in rheumatoid arthritis (RA) treatment and, to our knowledge, has never been reported in association with TCZ therapy. We herein report four agranulocytosis cases in three patients, with the first two cases suspected to be secondary to human parvovirus B19 (PVB19) infection. Agranulocytosis manifested in the first patient 2 months following a third RTX course. Bone marrow (BM) polymerase chain reaction (PCR) was positive for PVB19. The patient relapsed after three TCZ courses, with her PCR again positive for PVB19. Both episodes resolved under granulocyte-macrophage colony-stimulating factor (GM-CSF). In the second patient, agranulocytosis manifested after the 74th TCZ course. Bone marrow PCR was positive for PVB19, and the evolution was favorable under intravenous immunoglobulin administration. The third case was a 53-year-old female patient with seropositive RA who presented agranulocytosis after the first infusion of her fourth RTX course. Unfortunately, no PCR PVB19 was made on myelogram. Evolution was favorable after 5 days of GM-CSF. PVB19 infection should be investigated in patients suffering from agranulocytosis manifesting during biotherapy. In cases manifesting from the 15th day of RTX treatment onwards, hemogram must be conducted before readministering the infusion. PMID:27541023

  3. Prevalence of Hepatitis C Virus Genotypes in Mashhad, Northeast Iran

    PubMed Central

    Vossughinia, H; Goshayeshi, LA; Bayegi, H Rafatpanah; Sima, H; Kazemi, A; Erfani, S; Abedini, S; Goshayeshi, LE; Ghaffarzadegan, K; Nomani, H; Jamehdar, S Amel

    2012-01-01

    Background: Hepatitis C is a disease with significant global impact. The distribution of hepatitis C virus (HCV) genotypes in Mashhad (the Northeast and the biggest city after the capital of Iran) is unknown. The purpose of this study was to determine the prevalence of HCV genotypes among HCV seropositive patients, and to study the relationship between types, virologic and demographic features of patients in Mashhad. Methods: Three hundred and eighty-two clinical specimens obtained from HCV-infected patients referred to Ghaem Hospital in Mashhad during a period of 2009 to 2010 were selected. HCV genotype was determined by Nested PCR amplification of HCV core gene using genotype specific primers. Results: Totally, 299 patients were male (79.9%). The most common HCV genotype was genotype 3a, with 150 (40%) of subjects. Genotype 1a was the other frequent genotype, with 147(39.2%) subjects. Frequency of genotypes for 1b, 5 and 2 was 41(10.9%), 13(3.4%) and 9(2.4%), respectively. Mix genotype including 1a+1b in 4 (1.04%), 1a+3a in 3 (0.8%) was found in 7 patients. Four percent out of these samples had an undetermined genotype. Among the hemophilia patient, there were 13(48.1%) genotypes as 1a, 3(11.1%) 1b and 10(37%) 3a, respectively. Conclusion: The dominant HCV genotype among patients living in Mashhad was 3a. This study gives added evidence of the predominant HCV genotypes in Iran. PMID:23193507

  4. Axiom turkey genotyping array

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Axiom®Turkey Genotyping Array interrogates 643,845 probesets on the array, covering 643,845 SNPs. The array development was led by Dr. Julie Long of the USDA-ARS Beltsville Agricultural Research Center under a public-private partnership with Hendrix Genetics, Aviagen, and Affymetrix. The Turk...

  5. Bcl-2/adenovirus E1B 19 kDa interacting protein-3 knockdown enables growth of breast cancer metastases in the lung, liver, and bone.

    PubMed

    Manka, David; Spicer, Zachary; Millhorn, David E

    2005-12-15

    The mouse breast cancer cell lines 4T1, 4T07, and 67NR are highly tumorigenic but vary in metastatic potential: 4T1 widely disseminates, resulting in secondary tumors in the lung, liver, bone, and brain; 4T07 spreads to the lung and liver but is unable to establish metastatic nodules; 67NR is unable to metastasize. The Bcl-2/adenovirus E1B 19 kDa interacting protein-3 (Bnip-3) was recently shown to be absent after hypoxia in pancreatic cancer cell lines whereas its overexpression restored hypoxia-induced cell death. We found that Bnip-3 expression increased after 6 hours of hypoxia in all cell lines tested but was highest in the nonmetastatic 67NR cells and lowest in the highly metastatic 4T1 cells. Hypoxia-induced expression of Bnip-3 in the disseminating but nonmetastatic 4T07 cells was intermediate compared with 4T1 and 67NR cells. Cleaved caspase-3, a key downstream effector of cell death, increased after 6 hours of hypoxia in the 67NR and 4T07 cells by 1.9- and 2.5-fold, respectively. Conversely, cleaved caspase-3 decreased by 45% in the highly metastatic 4T1 cells after hypoxia. Small interfering RNA oligonucleotides targeting endogenous Bnip-3 blocked cell death and increased clonigenic survival after hypoxic challenge in vitro and increased primary tumor size and enabled metastasis to the lung, liver, and sternum of mice inoculated with 4T07 cells in vivo. These data inversely correlate the hypoxia-induced expression of the cell death protein Bnip-3 to metastatic potential and suggest that loss of Bnip-3 expression is critical for malignant and metastatic evasion of hypoxia-induced cell death. PMID:16357180

  6. Genomic Analysis of 15 Human Coronaviruses OC43 (HCoV-OC43s) Circulating in France from 2001 to 2013 Reveals a High Intra-Specific Diversity with New Recombinant Genotypes

    PubMed Central

    Kin, Nathalie; Miszczak, Fabien; Lin, Wei; Ar Gouilh, Meriadeg; Vabret, Astrid

    2015-01-01

    Human coronavirus OC43 (HCoV-OC43) is one of five currently circulating human coronaviruses responsible for respiratory infections. Like all coronaviruses, it is characterized by its genome’s high plasticity. The objectives of the current study were to detect genetically distinct genotypes and eventually recombinant genotypes in samples collected in Lower Normandy between 2001 and 2013. To this end, we sequenced complete nsp12, S, and N genes of 15 molecular isolates of HCoV-OC43 from clinical samples and compared them to available data from the USA, Belgium, and Hong-Kong. A new cluster E was invariably detected from nsp12, S, and N data while the analysis of nsp12 and N genes revealed the existence of new F and G clusters respectively. The association of these different clusters of genes in our specimens led to the description of thirteen genetically distinct genotypes, among which eight recombinant viruses were discovered. Identification of these recombinant viruses, together with temporal analysis and tMRCA estimation, provides important information for understanding the dynamics of the evolution of these epidemic coronaviruses. PMID:26008694

  7. HLA genotyping in pediatric celiac disease patients

    PubMed Central

    Stanković, Biljana; Radlović, Nedeljko; Leković, Zoran; Ristić, Dragana; Radlović, Vladimir; Nikčević, Gordana; Kotur, Nikola; Vučićević, Ksenija; Kostić, Tatjana; Pavlović, Sonja; Zukić, Branka

    2014-01-01

    Celiac disease (CD) is a chronic inflammatory disease in the small intestine triggered by gluten uptake that occurs in genetically susceptible individuals. HLA-DQ2 protein encoded by HLA-DQA1*05 and DQB1*02 alleles is found in 90-95% of CD patients. All of the remaining patients carry HLA-DQ8 protein encoded by HLA-DQA1*03 and DQB1*03:02 alleles. Specific HLA-DQ genotypes define different risk for CD incidence. Presence of susceptible HLA-DQ genotypes does not predict certain disease development, but their absence makes CD very unlikely, close to 100%. Here we presented for the first time the distribution of HLA-DQ genotypes in the group of pediatric celiac patients from the University Children’s Hospital, Belgrade, Serbia and estimated risk for CD development that these genotypes confer. Seventy three celiac disease patients and 62 healthy individuals underwent genotyping for DQA1, DQB1 alleles and DRB1 allele. 94.5% of patients carried alleles that encode DQ2 protein variant and 2.7% carried alleles that encode DQ8 protein variant. Two patients carried single DQB1*02 allele. No patients were negative for all the alleles predisposing to CD. The highest HLA-DQ genotype risk for CD development was found in group of patients homozygous for DQ2.5 haplotype, followed by the group of heterozygous carriers of DQ2.5 haplotype in combination with DQB1*02 allele within the other haplotype. The lowest risk was observed in carriers of a single copy of DQB1*02 or DQA1*05 allele or other non-predisposing alleles. HLA genotyping, more informative than serological testing commonly used, proved to be a useful diagnostic tool for excluding CD development. PMID:25172978

  8. Precise genotyping and recombination detection of Enterovirus.

    PubMed

    Lin, Chieh-Hua; Wang, Yu-Bin; Chen, Shu-Hwa; Hsiung, Chao Agnes; Lin, Chung-Yen

    2015-01-01

    Enteroviruses (EV) with different genotypes cause diverse infectious diseases in humans and mammals. A correct EV typing result is crucial for effective medical treatment and disease control; however, the emergence of novel viral strains has impaired the performance of available diagnostic tools. Here, we present a web-based tool, named EVIDENCE (EnteroVirus In DEep conception, http://symbiont.iis.sinica.edu.tw/evidence), for EV genotyping and recombination detection. We introduce the idea of using mixed-ranking scores to evaluate the fitness of prototypes based on relatedness and on the genome regions of interest. Using phylogenetic methods, the most possible genotype is determined based on the closest neighbor among the selected references. To detect possible recombination events, EVIDENCE calculates the sequence distance and phylogenetic relationship among sequences of all sliding windows scanning over the whole genome. Detected recombination events are plotted in an interactive figure for viewing of fine details. In addition, all EV sequences available in GenBank were collected and revised using the latest classification and nomenclature of EV in EVIDENCE. These sequences are built into the database and are retrieved in an indexed catalog, or can be searched for by keywords or by sequence similarity. EVIDENCE is the first web-based tool containing pipelines for genotyping and recombination detection, with updated, built-in, and complete reference sequences to improve sensitivity and specificity. The use of EVIDENCE can accelerate genotype identification, aiding clinical diagnosis and enhancing our understanding of EV evolution.

  9. Precise genotyping and recombination detection of Enterovirus

    PubMed Central

    2015-01-01

    Enteroviruses (EV) with different genotypes cause diverse infectious diseases in humans and mammals. A correct EV typing result is crucial for effective medical treatment and disease control; however, the emergence of novel viral strains has impaired the performance of available diagnostic tools. Here, we present a web-based tool, named EVIDENCE (EnteroVirus In DEep conception, http://symbiont.iis.sinica.edu.tw/evidence), for EV genotyping and recombination detection. We introduce the idea of using mixed-ranking scores to evaluate the fitness of prototypes based on relatedness and on the genome regions of interest. Using phylogenetic methods, the most possible genotype is determined based on the closest neighbor among the selected references. To detect possible recombination events, EVIDENCE calculates the sequence distance and phylogenetic relationship among sequences of all sliding windows scanning over the whole genome. Detected recombination events are plotted in an interactive figure for viewing of fine details. In addition, all EV sequences available in GenBank were collected and revised using the latest classification and nomenclature of EV in EVIDENCE. These sequences are built into the database and are retrieved in an indexed catalog, or can be searched for by keywords or by sequence similarity. EVIDENCE is the first web-based tool containing pipelines for genotyping and recombination detection, with updated, built-in, and complete reference sequences to improve sensitivity and specificity. The use of EVIDENCE can accelerate genotype identification, aiding clinical diagnosis and enhancing our understanding of EV evolution. PMID:26678286

  10. Precise genotyping and recombination detection of Enterovirus.

    PubMed

    Lin, Chieh-Hua; Wang, Yu-Bin; Chen, Shu-Hwa; Hsiung, Chao Agnes; Lin, Chung-Yen

    2015-01-01

    Enteroviruses (EV) with different genotypes cause diverse infectious diseases in humans and mammals. A correct EV typing result is crucial for effective medical treatment and disease control; however, the emergence of novel viral strains has impaired the performance of available diagnostic tools. Here, we present a web-based tool, named EVIDENCE (EnteroVirus In DEep conception, http://symbiont.iis.sinica.edu.tw/evidence), for EV genotyping and recombination detection. We introduce the idea of using mixed-ranking scores to evaluate the fitness of prototypes based on relatedness and on the genome regions of interest. Using phylogenetic methods, the most possible genotype is determined based on the closest neighbor among the selected references. To detect possible recombination events, EVIDENCE calculates the sequence distance and phylogenetic relationship among sequences of all sliding windows scanning over the whole genome. Detected recombination events are plotted in an interactive figure for viewing of fine details. In addition, all EV sequences available in GenBank were collected and revised using the latest classification and nomenclature of EV in EVIDENCE. These sequences are built into the database and are retrieved in an indexed catalog, or can be searched for by keywords or by sequence similarity. EVIDENCE is the first web-based tool containing pipelines for genotyping and recombination detection, with updated, built-in, and complete reference sequences to improve sensitivity and specificity. The use of EVIDENCE can accelerate genotype identification, aiding clinical diagnosis and enhancing our understanding of EV evolution. PMID:26678286

  11. PCR-based approach to distinguish group A human rotavirus genotype 1 vs. genotype 2 genes.

    PubMed

    McKell, Allison O; Nichols, Joshua C; McDonald, Sarah M

    2013-12-01

    Group A rotaviruses (RVs) are eleven-segmented, double-stranded RNA viruses and important causes of severe diarrhea in children. A full-genome classification system is readily used to describe the genetic makeup of individual RV strains. In this system, each viral gene is assigned a specific genotype based upon its nucleotide sequence and established percent identity cut-off values. However, a faster and more cost-effective approach to determine RV gene genotypes is to utilize specific oligonucleotide primer sets in RT-PCR/PCR. Such primer sets and PCR-based genotyping methods have already been developed for the VP7-, VP6-, VP4- and NSP4-coding gene segments. In this study, primers were developed for the remaining seven RV gene segments, which encode proteins VP1, VP2, VP3, NSP1, NSP2, NSP3, and NSP5/6. Specifically, primers were designed to distinguish the two most common human RV genotypes (1 vs. 2) for these genes and were validated on several cell culture-adapted human and animal RV strains, as well as on human RVs from clinical fecal specimens. As such, primer sets now exist for all eleven genes of common human RVs, allowing for the identification of reassortant strains with mixed constellations of both genotype 1 and 2 genes using a rapid and economical RT-PCR/PCR method. PMID:24012969

  12. Genomic Variants Revealed by Invariably Missing Genotypes in Nelore Cattle

    PubMed Central

    da Silva, Joaquim Manoel; Giachetto, Poliana Fernanda; da Silva, Luiz Otávio Campos; Cintra, Leandro Carrijo; Paiva, Samuel Rezende; Caetano, Alexandre Rodrigues; Yamagishi, Michel Eduardo Beleza

    2015-01-01

    High density genotyping panels have been used in a wide range of applications. From population genetics to genome-wide association studies, this technology still offers the lowest cost and the most consistent solution for generating SNP data. However, in spite of the application, part of the generated data is always discarded from final datasets based on quality control criteria used to remove unreliable markers. Some discarded data consists of markers that failed to generate genotypes, labeled as missing genotypes. A subset of missing genotypes that occur in the whole population under study may be caused by technical issues but can also be explained by the presence of genomic variations that are in the vicinity of the assayed SNP and that prevent genotyping probes from annealing. The latter case may contain relevant information because these missing genotypes might be used to identify population-specific genomic variants. In order to assess which case is more prevalent, we used Illumina HD Bovine chip genotypes from 1,709 Nelore (Bos indicus) samples. We found 3,200 missing genotypes among the whole population. NGS re-sequencing data from 8 sires were used to verify the presence of genomic variations within their flanking regions in 81.56% of these missing genotypes. Furthermore, we discovered 3,300 novel SNPs/Indels, 31% of which are located in genes that may affect traits of importance for the genetic improvement of cattle production. PMID:26305794

  13. Trichothecene genotypes of Fusarium graminearum from wheat in Uruguay.

    PubMed

    Pan, Dinorah; Calero, Natalia; Mionetto, Ana; Bettucci, Lina

    2013-03-01

    Gibberella zeae (Schwein.) Petch (anamorph F. graminearum Schwabe) is the primary causal agent of FHB of wheat in Uruguay. In the last decade, F. graminearum has produced destructive epidemics on wheat in Uruguay, causing yield losses and price discounts due to reduced seed quality. Strains of F. graminearum clade usually express one of three strain-specific profiles of trichothecene metabolites: nivalenol and its acetylated derivatives (NIV chemotype), deoxynivalenol and 3-acetyldeoxynivalenol (3-AcDON chemotype), or deoxynivalenol and 15-acetyldeoxynivalenol (15-AcDON chemotype). A multiplex PCR assay of Tri3, Tri5, and Tri7 was used to determine the trichothecene genotype of 111 strains of F. graminearum collected during 2003 and 2009 growing seasons from fields located in the major wheat production area of Uruguay. The result showed that all except one of the isolates were of DON genotype, with the remainder of NIV genotype in years 2003 and 2009. All strains with the DON genotype were also of the 15-AcDON genotype in 2003 and nearly all (45/50) in 2009. No DON/3-AcDON genotypes were found in either growing season. No potential shifts in the populations were found in the trichothecene genotypes between 2003 and the 2009 epidemic FHB harvest seasons. This study provides the first data on trichothecene genotypes of F. graminearum strains isolated from wheat in Uruguay and add to the current regional knowledge of trichothecene genotypes.

  14. Genomic Variants Revealed by Invariably Missing Genotypes in Nelore Cattle.

    PubMed

    da Silva, Joaquim Manoel; Giachetto, Poliana Fernanda; da Silva, Luiz Otávio Campos; Cintra, Leandro Carrijo; Paiva, Samuel Rezende; Caetano, Alexandre Rodrigues; Yamagishi, Michel Eduardo Beleza

    2015-01-01

    High density genotyping panels have been used in a wide range of applications. From population genetics to genome-wide association studies, this technology still offers the lowest cost and the most consistent solution for generating SNP data. However, in spite of the application, part of the generated data is always discarded from final datasets based on quality control criteria used to remove unreliable markers. Some discarded data consists of markers that failed to generate genotypes, labeled as missing genotypes. A subset of missing genotypes that occur in the whole population under study may be caused by technical issues but can also be explained by the presence of genomic variations that are in the vicinity of the assayed SNP and that prevent genotyping probes from annealing. The latter case may contain relevant information because these missing genotypes might be used to identify population-specific genomic variants. In order to assess which case is more prevalent, we used Illumina HD Bovine chip genotypes from 1,709 Nelore (Bos indicus) samples. We found 3,200 missing genotypes among the whole population. NGS re-sequencing data from 8 sires were used to verify the presence of genomic variations within their flanking regions in 81.56% of these missing genotypes. Furthermore, we discovered 3,300 novel SNPs/Indels, 31% of which are located in genes that may affect traits of importance for the genetic improvement of cattle production. PMID:26305794

  15. Susceptibility of biallelic haplotype and genotype frequencies to genotyping error.

    PubMed

    Moskvina, Valentina; Schmidt, Karl Michael

    2006-12-01

    With the availability of fast genotyping methods and genomic databases, the search for statistical association of single nucleotide polymorphisms with a complex trait has become an important methodology in medical genetics. However, even fairly rare errors occurring during the genotyping process can lead to spurious association results and decrease in statistical power. We develop a systematic approach to study how genotyping errors change the genotype distribution in a sample. The general M-marker case is reduced to that of a single-marker locus by recognizing the underlying tensor-product structure of the error matrix. Both method and general conclusions apply to the general error model; we give detailed results for allele-based errors of size depending both on the marker locus and the allele present. Multiple errors are treated in terms of the associated diffusion process on the space of genotype distributions. We find that certain genotype and haplotype distributions remain unchanged under genotyping errors, and that genotyping errors generally render the distribution more similar to the stable one. In case-control association studies, this will lead to loss of statistical power for nondifferential genotyping errors and increase in type I error for differential genotyping errors. Moreover, we show that allele-based genotyping errors do not disturb Hardy-Weinberg equilibrium in the genotype distribution. In this setting we also identify maximally affected distributions. As they correspond to situations with rare alleles and marker loci in high linkage disequilibrium, careful checking for genotyping errors is advisable when significant association based on such alleles/haplotypes is observed in association studies.

  16. Dysregulation Of Innate Immunity In HCV Genotype 1 IL28B Unfavorable Genotype Patients: Impaired Viral Kinetics And Therapeutic Response

    PubMed Central

    Naggie, Susanna; Osinusi, Anu; Katsounas, Antonios; Lempicki, Richard; Herrmann, Eva; Thompson, Alexander J; Clark, Paul J; Patel, Keyur; Muir, Andrew J; McHutchison, John G; Schlaak, Joerg F; Trippler, Martin; Shivakumar, Bhavana; Masur, Henry; Polis, Michael A.; Kottilil, Shyam

    2012-01-01

    Recent studies have shown that a single nucleotide polymorphism upstream of the interleukin (IL)-28B gene plays a major role in predicting therapeutic response in HCV-infected patients treated with pegylated interferon-alpha (IFN)/ribavirin. We sought to investigate the mechanism of the IL28B polymorphism, specifically as it relates to early HCV viral kinetics (VK), IFN pharmacokinetics (PK), IFN pharmacodynamics (PD), and gene expression profiles. Two prospective cohorts (HIV/HCV co-infected and HCV mono-infected) completing treatment with IFN/ribavirin were enrolled. Patients (N=88; 44 HIV/HCV and 44 HCV) were genotyped at the polymorphic site rs12979860. In the HIV/HCV cohort frequent serum sampling was completed for HCV RNA and IFN-levels. DNA microarray of PBMCs and individual expression of interferon stimulated genes (ISGs) were quantified on IFN-therapy. The IL28B favorable (CC) genotype was associated with improved therapeutic response compared to unfavorable (CT/TT) genotypes. Patients with favorable genotype had greater first and second phase VK (P=0.004 and P=0.036, respectively), IFN maximum anti-viral efficiency (P=0.007) and infected cell death loss (P=0.009) compared to unfavorable genotypes. Functional annotation analysis of DNA microarray data was consistent with depressed innate immune function, particularly of NK cells, from patients with unfavorable genotypes (P<0.004). Induction of innate immunity genes was also lower in unfavorable genotype. ISG expression at baseline and induction with IFN was independent of IL28B genotype. Conclusions Carriers of the IL28B favorable genotype were more likely to have superior innate immune response to IFN-therapy compared to unfavorable genotypes, this suggests the unfavorable genotype has aberrant baseline induction of innate immune response pathways resulting in impaired virologic response. IL28B genotype is associated with more rapid viral kinetics and improved treatment response outcomes independent of

  17. Genotypes of hepatitis B virus among voluntary blood donors in northern Thailand.

    PubMed

    Jutavijittum, Prapan; Jiviriyawat, Yupa; Yousukh, Amnat; Kunachiwa, Warunee; Toriyama, Kan

    2006-08-01

    There are distinct ethnogeographic variations for the distribution of various hepatitis B virus (HBV) genotypes, and pathogenic and therapeutic differences are also observed. In general, genotype B infection has a relatively better prognosis than genotype C. In Thailand, genotypes C and B were reported as the major genotypes; however, there were no previous reports of HBV genotyping in the north of the country. From 1998 to 2000, 216 HBsAg-positive serum samples (164 males and 52 females, aged 16-52 years), were screened and collected from voluntary blood donors in four provinces of northern Thailand. The method of Naito et al. was employed in this study, with the multiplex-PCR approach and genotype-specific primers to identify genotypes A-F. We found that the HBV genotype C was highly predominant in northern Thailand (89.3%), when compared with the previous reports of genotype C distribution among voluntary blood donors from other areas in the country (50-65%), followed by genotype B (7.4%), mixed infection of genotype B and C (1.9%) and genotype A (0.5%). Four samples (1.9%) were unclassifiable. There was no significant difference of genotype distribution among four northern Thai provinces or each age group.

  18. In-situ X-ray diffraction studies of the phase transformations and structural states of B2, R and B19′ phases in Ti{sub 49.5}Ni{sub 50.5} alloy

    SciTech Connect

    Ostapenko, Marina G.; Meisner, Ludmila L.; Lotkov, Aleksandr I. Gudimova, Ekaterina Y.; Zakharova, Margarita A.

    2015-10-27

    The martensitic transformation, Debye–Waller factor, mean-square atomic displacements and the coefficient of thermal expansion on cooling of the Ti{sub 49.5}Ni{sub 50.5} shape memory alloy were examined using in-situ X-ray diffraction. It was revealed B2→R (T{sub R} ≡ T = 273 ± 10 K) along with B2→B19’ (M{sub s} ≡ T = 273 ± 10 K) transitions occur. It was found that Debye–Waller factor and mean-square displacement of B2 phase undergo significant increase as functions of temperature when phase transition B2→R and B2→B19’ take place. The analysis of the thermal expansion coefficient of the B2 phase indicates that the value of a increases almost linearly while cooling.

  19. Genotyping Sleep Disorders Patients

    PubMed Central

    Shadan, Farhad F.; Dawson, Arthur; Cronin, John W.; Jamil, Shazia M.; Grizas, Alexandra P.; Koziol, James A.; Kline, Lawrence E.

    2010-01-01

    Objective The genetic susceptibility factors underlying sleep disorders might help us predict prognoses and responses to treatment. Several candidate polymorphisms for sleep disorders have been proposed, but there has as yet inadequate replication or validation that the candidates may be useful in the clinical setting. Methods To assess the validity of several candidate associations, we obtained saliva deoxyribonucleic acid (DNA) samples and clinical information from 360 consenting research participants who were undergoing clinical polysomnograms. Ten single nucleotide polymorphisms (SNPs) were genotyped. These were thought to be related to depression, circadian sleep disorders, sleep apnea, restless legs syndrome (RLS), excessive sleepiness, or to slow waves in sleep. Results With multivariate generalized linear models, the association of TEF rs738499 with depressive symptoms was confirmed. Equivocal statistical evidence of association of rs1801260 (the C3111T SNP in the CLOCK gene) with morningness/eveningness and an association of Apolipoprotein E (APOE) rs429358 with the Epworth Sleepiness Scale (ESS) were obtained, but these associations were not strong enough to be of clinical value by themselves. Predicted association of SNPs with sleep apnea, RLS, and slow wave sleep were not confirmed. Conclusion The SNPs tested would not, by themselves, be of use for clinical genotyping in a sleep clinic. PMID:20396431

  20. Pattern and molecular epidemiology of Hepatitis B virus genotypes circulating in Pakistan.

    PubMed

    Awan, Zunaira; Idrees, Muhammad; Amin, Irum; Butt, Sadia; Afzal, Samia; Akbar, Haji; Rehman, Irshad-ur; Younas, Saima; Shahid, Muhammad; Lal, Amreek; Saleem, Sana; Rauff, Bisma

    2010-12-01

    The continuously mutating nature of Hepatitis B virus (HBV) is responsible for the emergence of varying genotypes in different regions of the world affecting the disease outcome. The objective of the current study was to find out the pattern of HBV genotypes circulating in Pakistan. HBV genotypes were determined in HBV chronic patients of different age and gender from all the four different geographical regions (provinces) of Pakistan for a period of 2 years (2007-2009). Out of the total 3137 consecutive patients, 300 (175; 58.3% males and 125; 41.7% females) were randomly selected for HBV genotype A through H determination using molecular genotyping methods. Total 269 (89.6%) isolates were successfully genotyped where as 31 (10.3%) samples failed to generate a type-specific PCR band and were found untypable. Out of the successfully genotyped samples, 43 (14.3%) were with type A, 54 (18%) were with type B, 83 (27.6%) were with type C, 39 (13%) were with type D, 2 (0.6%) were with type E, 4 (1.3%) were with genotype F and total 44 (14.6%) were with mixed HBV infections. Of the mixed genotype infection cases, 16 were with genotypes A/D, 9 were B/C, six were A/D/F, five were with genotypes A/F, two were with A/B/D and B/E and one each for A/C as well as A/E genotypes. Four common genotypes of HBV found worldwide (A, B, C & D) were isolated from Pakistan along with uncommon genotypes E and F for the first time in Pakistan. Overall Genotype C is the most prevalent genotype. Genotypes B and C are predominant in Punjab & Balochistan and Khyber Pakhtoonkhwa, respectively whereas genotype A in Sindh.

  1. Newcastle Disease Virus in Madagascar: Identification of an Original Genotype Possibly Deriving from a Died Out Ancestor of Genotype IV

    PubMed Central

    Maminiaina, Olivier F.; Gil, Patricia; Briand, François-Xavier; Albina, Emmanuel; Keita, Djénéba; Andriamanivo, Harentsoaniaina Rasamoelina; Chevalier, Véronique; Lancelot, Renaud; Martinez, Dominique; Rakotondravao, R.; Rajaonarison, Jean-Joseph; Koko, M.; Andriantsimahavandy, Abel A.; Jestin, Véronique; Servan de Almeida, Renata

    2010-01-01

    In Madagascar, Newcastle disease (ND) has become enzootic after the first documented epizootics in 1946, with recurrent annual outbreaks causing mortality up to 40%. Four ND viruses recently isolated in Madagascar were genotypically and pathotypically characterised. By phylogenetic inference based on the F and HN genes, and also full-genome sequence analyses, the NDV Malagasy isolates form a cluster distant enough to constitute a new genotype hereby proposed as genotype XI. This new genotype is presumably deriving from an ancestor close to genotype IV introduced in the island probably more than 50 years ago. Our data show also that all the previously described neutralising epitopes are conserved between Malagasy and vaccine strains. However, the potential implication in vaccination failures of specific amino acid substitutions predominantly found on surface-exposed epitopes of F and HN proteins is discussed. PMID:21085573

  2. Serotypes and genotypes of invasive pneumococci in the central part of Slovenia.

    PubMed

    Müller Premru, Manica; Beović, Bojana; Pokorn, Marko; Cvitković Špik, Vesna

    2015-09-01

    To investigate epidemiology of invasive pneumococcal disease (IPD) in the central part of Slovenia in a population with no routine pneumococcal vaccination, we carried out serotyping of isolates by sequential multiplex polymerase chain reaction (PCR) and genotyping by repetitive sequence-based PCR (rep-PCR) and some by multilocus sequence typing. IPD was confirmed in 134 (26.5 %) of 510 acutely ill patients, either by a positive blood culture or real-time PCR (rt-PCR). In 94 patients, isolates were available for typing (24 from blood and 70 from nasopharynx). They belonged to 12 different serotypes; the most prevalent were 14 (27.6 % isolates), 9V, 3 (12.7 % each), 7F (9.5 %), 19A, and 1 (7.4 % each) followed by 4, 6A/B, 19F, 23F, 18C, and 33F. Genotyping yielded 34 rep-PCR genotypes and 13 subtypes; six were found in serotype 14, one in 9V, four each in 3, 19A, and 6A/B, three each in 7F and 1, and two each in 4, 19F, 23F, and 18C. Serotype 9V was the most homogenous and 14 and 19A were heterogenous and had two divergent clonal groups each. The most common genotypes belonged to virulent widespread clones, like ST162, ST9, ST15, ST156, ST191, and ST1377; however, sporadic clones were also observed.

  3. Genotyping of Coxiella burnetii from domestic ruminants and human in Hungary: indication of various genotypes

    PubMed Central

    2014-01-01

    Background Information about the genotypic characteristic of Coxiella burnetii from Hungary is lacking. The aim of this study is to describe the genetic diversity of C. burnetii in Hungary and compare genotypes with those found elsewhere. A total of 12 samples: (cattle, n = 6, sheep, n = 5 and human, n = 1) collected from across Hungary were studied by a 10-loci multispacer sequence typing (MST) and 6-loci multiple-locus variable-number of tandem repeat analysis (MLVA). Phylogenetic relationships among MST genotypes show how these Hungarian samples are related to others collected around the world. Results Three MST genotypes were identified: sequence type (ST) 20 has also been identified in ruminants from other European countries and the USA, ST28 was previously identified in Kazakhstan, and the proposed ST37 is novel. All MST genotypes yielded different MLVA genotypes and three different MLVA genotypes were identified within ST20 samples alone. Two novel MLVA types 0-9-5-5-6-2 (AG) and 0-8-4-5-6-2 (AF) (Ms23-Ms24-Ms27-Ms28-Ms33-Ms34) were defined in the ovine materials correlated with ST28 and ST37. Samples from different parts of the phylogenetic tree were associated with different hosts, suggesting host-specific adaptations. Conclusions Even with the limited number of samples analysed, this study revealed high genetic diversity among C. burnetii in Hungary. Understanding the background genetic diversity will be essential in identifying and controlling outbreaks. PMID:24885415

  4. HBV Genotypic Variability in Cuba

    PubMed Central

    Loureiro, Carmen L.; Aguilar, Julio C.; Aguiar, Jorge; Muzio, Verena; Pentón, Eduardo; Garcia, Daymir; Guillen, Gerardo; Pujol, Flor H.

    2015-01-01

    The genetic diversity of HBV in human population is often a reflection of its genetic admixture. The aim of this study was to explore the genotypic diversity of HBV in Cuba. The S genomic region of Cuban HBV isolates was sequenced and for selected isolates the complete genome or precore-core sequence was analyzed. The most frequent genotype was A (167/250, 67%), mainly A2 (149, 60%) but also A1 and one A4. A total of 77 isolates were classified as genotype D (31%), with co-circulation of several subgenotypes (56 D4, 2 D1, 5 D2, 7 D3/6 and 7 D7). Three isolates belonged to genotype E, two to H and one to B3. Complete genome sequence analysis of selected isolates confirmed the phylogenetic analysis performed with the S region. Mutations or polymorphisms in precore region were more common among genotype D compared to genotype A isolates. The HBV genotypic distribution in this Caribbean island correlates with the Y lineage genetic background of the population, where a European and African origin prevails. HBV genotypes E, B3 and H isolates might represent more recent introductions. PMID:25742179

  5. HBV genotypic variability in Cuba.

    PubMed

    Loureiro, Carmen L; Aguilar, Julio C; Aguiar, Jorge; Muzio, Verena; Pentón, Eduardo; Garcia, Daymir; Guillen, Gerardo; Pujol, Flor H

    2015-01-01

    The genetic diversity of HBV in human population is often a reflection of its genetic admixture. The aim of this study was to explore the genotypic diversity of HBV in Cuba. The S genomic region of Cuban HBV isolates was sequenced and for selected isolates the complete genome or precore-core sequence was analyzed. The most frequent genotype was A (167/250, 67%), mainly A2 (149, 60%) but also A1 and one A4. A total of 77 isolates were classified as genotype D (31%), with co-circulation of several subgenotypes (56 D4, 2 D1, 5 D2, 7 D3/6 and 7 D7). Three isolates belonged to genotype E, two to H and one to B3. Complete genome sequence analysis of selected isolates confirmed the phylogenetic analysis performed with the S region. Mutations or polymorphisms in precore region were more common among genotype D compared to genotype A isolates. The HBV genotypic distribution in this Caribbean island correlates with the Y lineage genetic background of the population, where a European and African origin prevails. HBV genotypes E, B3 and H isolates might represent more recent introductions.

  6. Genotyping-in-Thousands by sequencing (GT-seq): A cost effective SNP genotyping method based on custom amplicon sequencing.

    PubMed

    Campbell, Nathan R; Harmon, Stephanie A; Narum, Shawn R

    2015-07-01

    Genotyping-in-Thousands by sequencing (GT-seq) is a method that uses next-generation sequencing of multiplexed PCR products to generate genotypes from relatively small panels (50-500) of targeted single-nucleotide polymorphisms (SNPs) for thousands of individuals in a single Illumina HiSeq lane. This method uses only unlabelled oligos and PCR master mix in two thermal cycling steps for amplification of targeted SNP loci. During this process, sequencing adapters and dual barcode sequence tags are incorporated into the amplicons enabling thousands of individuals to be pooled into a single sequencing library. Post sequencing, reads from individual samples are split into individual files using their unique combination of barcode sequences. Genotyping is performed with a simple perl script which counts amplicon-specific sequences for each allele, and allele ratios are used to determine the genotypes. We demonstrate this technique by genotyping 2068 individual steelhead trout (Oncorhynchus mykiss) samples with a set of 192 SNP markers in a single library sequenced in a single Illumina HiSeq lane. Genotype data were 99.9% concordant to previously collected TaqMan(™) genotypes at the same 192 loci, but call rates were slightly lower with GT-seq (96.4%) relative to Taqman (99.0%). Of the 192 SNPs, 187 were genotyped in ≥90% of the individual samples and only 3 SNPs were genotyped in <70% of samples. This study demonstrates amplicon sequencing with GT-seq greatly reduces the cost of genotyping hundreds of targeted SNPs relative to existing methods by utilizing a simple library preparation method and massive efficiency of scale.

  7. Greater diversity of hepatitis C virus genotypes found in Hong Kong than in mainland China.

    PubMed Central

    Zhang, Y Y; Lok, A S; Chan, D T; Widell, A

    1995-01-01

    A hepatitis C virus (HCV) genotyping PCR assay based on type-specific primers was expanded to include genotype 6a as well as genotypes 1a, 1b, 2a, and 3a. The nucleotide sequences of a 194-bp fragment in the center of the HCV core gene showed that the homologies between genotype 6a and genotypes 1a, 1b, 2a, 2b, 3a, and 5 were 81.2, 82.1, 73.8, 77.3, 81.4, and 78.9%, respectively. A high degree of homology (99.6%) was seen in the amplified core region among eight clinically unrelated genotype 6a isolates. Although the Hong Kong Chinese patients had predominantly genotype 1b (70%), it was noteworthy that genotype 6a was the second most common genotype (14%). Four other HCV genotypes--1a, 1b, 2a, and 2b--were also present. In contrast, HCV infection by mainland China was confined to genotypes 1b and 2a. Thus, we found a greater diversity of HCV genotypes in Hong Kong than in mainland China. PMID:8576348

  8. Prior infection of pigs with a genotype 3 swine hepatitis E virus (HEV) protects against subsequent challenges with homologous and heterologous genotypes 3 and 4 human HEV.

    PubMed

    Sanford, Brenton J; Dryman, Barbara A; Huang, Yao-Wei; Feagins, Alicia R; Leroith, Tanya; Meng, Xiang-Jin

    2011-07-01

    Hepatitis E virus (HEV) is an important human pathogen. At least four recognized and two putative genotypes of mammalian HEV have been reported: genotypes 1 and 2 are restricted to humans whereas genotypes 3 and 4 are zoonotic. The current experimental vaccines are all based on a single strain of HEV, even though multiple genotypes of HEV are co-circulating in some countries and thus an individual may be exposed to more than one genotype. Genotypes 3 and 4 swine HEV is widespread in pigs and known to infect humans. Therefore, it is important to know if prior infection with a genotype 3 swine HEV will confer protective immunity against subsequent exposure to genotypes 3 and 4 human and swine HEV. In this study, specific-pathogen-free pigs were divided into 4 groups of 6 each. Pigs in the three treatment groups were each inoculated with a genotype 3 swine HEV, and 12 weeks later, challenged with the same genotype 3 swine HEV, a genotype 3 human HEV, and a genotype 4 human HEV, respectively. The control group was inoculated and challenged with PBS buffer. Weekly sera from all pigs were tested for HEV RNA and IgG anti-HEV, and weekly fecal samples were also tested for HEV RNA. The pigs inoculated with swine HEV became infected as evidenced by fecal virus shedding and viremia, and the majority of pigs also developed IgG anti-HEV prior to challenge at 12 weeks post-inoculation. After challenge, viremia was not detected and only two pigs challenged with swine HEV had 1-week fecal virus shedding, suggesting that prior infection with a genotype 3 swine HEV prevented pigs from developing viremia and fecal virus shedding after challenges with homologous and heterologous genotypes 3 and 4 HEV. The results from this study have important implications for future development of an effective HEV vaccine.

  9. Special features of RAD Sequencing data: implications for genotyping

    PubMed Central

    Davey, John W; Cezard, Timothée; Fuentes-Utrilla, Pablo; Eland, Cathlene; Gharbi, Karim; Blaxter, Mark L

    2013-01-01

    Restriction site-associated DNA Sequencing (RAD-Seq) is an economical and efficient method for SNP discovery and genotyping. As with other sequencing-by-synthesis methods, RAD-Seq produces stochastic count data and requires sensitive analysis to develop or genotype markers accurately. We show that there are several sources of bias specific to RAD-Seq that are not explicitly addressed by current genotyping tools, namely restriction fragment bias, restriction site heterozygosity and PCR GC content bias. We explore the performance of existing analysis tools given these biases and discuss approaches to limiting or handling biases in RAD-Seq data. While these biases need to be taken seriously, we believe RAD loci affected by them can be excluded or processed with relative ease in most cases and that most RAD loci will be accurately genotyped by existing tools. PMID:23110438

  10. Special features of RAD Sequencing data: implications for genotyping.

    PubMed

    Davey, John W; Cezard, Timothée; Fuentes-Utrilla, Pablo; Eland, Cathlene; Gharbi, Karim; Blaxter, Mark L

    2013-06-01

    Restriction site-associated DNA Sequencing (RAD-Seq) is an economical and efficient method for SNP discovery and genotyping. As with other sequencing-by-synthesis methods, RAD-Seq produces stochastic count data and requires sensitive analysis to develop or genotype markers accurately. We show that there are several sources of bias specific to RAD-Seq that are not explicitly addressed by current genotyping tools, namely restriction fragment bias, restriction site heterozygosity and PCR GC content bias. We explore the performance of existing analysis tools given these biases and discuss approaches to limiting or handling biases in RAD-Seq data. While these biases need to be taken seriously, we believe RAD loci affected by them can be excluded or processed with relative ease in most cases and that most RAD loci will be accurately genotyped by existing tools.

  11. MassARRAY Spectrometry Is More Sensitive than PreTect HPV-Proofer and Consensus PCR for Type-Specific Detection of High-Risk Oncogenic Human Papillomavirus Genotypes in Cervical Cancer▿

    PubMed Central

    Basu, Partha; Chandna, Puneet; Bamezai, R. N. K.; Siddiqi, Maqsood; Saranath, Dhananjaya; Lear, Adrian; Ratnam, Sam

    2011-01-01

    Type-specific detection of human papillomavirus (HPV) is indicated for better risk stratification and clinical management of women testing positive for HPV and for epidemiologic surveillance. MassARRAY spectrometry (MassARRAY; Sequenom) is a novel method for type-specific detection of 15 high-risk oncogenic HPV types: HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -68, and -73. PreTect HPV-Proofer (Proofer; Norchip) is a type-specific assay that detects E6/E7 mRNA from five high-risk oncogenic HPV types: HPV-16, -18, -31, -33, and -45. The performance of these tests for type-specific identification of HPV was assessed with cervical specimens from 192 cases of cervical cancer in comparison with consensus MY09/MY11 PCR followed by nucleotide sequencing (consensus PCR). The overall HPV detection rates were 94.8% (95% confidence interval [CI], 91.7, 97.9), 83.3% (95% CI, 78.1, 88.5), and 86.5% (95% CI, 81.7, 91.3) for MassARRAY, Proofer, and consensus PCR, respectively. All tests were negative in six (3.1%) of the 192 cases. Considering only the specimens that contained at least one of the five types targeted by Proofer, the detection rates were 96.6%, 91.4%, and 86.9% for MassARRAY, Proofer, and consensus PCR, respectively. MassARRAY detected multiple infections in 14.1%, Proofer detected multiple infections in 3.6%, and consensus PCR failed to detect any multiple infections. The agreement was highest at 86.0% (kappa = 0.76) between MassARRAY and Proofer and lowest at 81.8% (kappa = 0.69) between Proofer and consensus PCR. In conclusion, MassARRAY is a highly sensitive and accurate method for type-specific detection of oncogenic HPV in cervical cancer, with Proofer showing impressive performance. PMID:21813716

  12. MassARRAY spectrometry is more sensitive than PreTect HPV-Proofer and consensus PCR for type-specific detection of high-risk oncogenic human papillomavirus genotypes in cervical cancer.

    PubMed

    Basu, Partha; Chandna, Puneet; Bamezai, R N K; Siddiqi, Maqsood; Saranath, Dhananjaya; Lear, Adrian; Ratnam, Sam

    2011-10-01

    Type-specific detection of human papillomavirus (HPV) is indicated for better risk stratification and clinical management of women testing positive for HPV and for epidemiologic surveillance. MassARRAY spectrometry (MassARRAY; Sequenom) is a novel method for type-specific detection of 15 high-risk oncogenic HPV types: HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -68, and -73. PreTect HPV-Proofer (Proofer; Norchip) is a type-specific assay that detects E6/E7 mRNA from five high-risk oncogenic HPV types: HPV-16, -18, -31, -33, and -45. The performance of these tests for type-specific identification of HPV was assessed with cervical specimens from 192 cases of cervical cancer in comparison with consensus MY09/MY11 PCR followed by nucleotide sequencing (consensus PCR). The overall HPV detection rates were 94.8% (95% confidence interval [CI], 91.7, 97.9), 83.3% (95% CI, 78.1, 88.5), and 86.5% (95% CI, 81.7, 91.3) for MassARRAY, Proofer, and consensus PCR, respectively. All tests were negative in six (3.1%) of the 192 cases. Considering only the specimens that contained at least one of the five types targeted by Proofer, the detection rates were 96.6%, 91.4%, and 86.9% for MassARRAY, Proofer, and consensus PCR, respectively. MassARRAY detected multiple infections in 14.1%, Proofer detected multiple infections in 3.6%, and consensus PCR failed to detect any multiple infections. The agreement was highest at 86.0% (kappa = 0.76) between MassARRAY and Proofer and lowest at 81.8% (kappa = 0.69) between Proofer and consensus PCR. In conclusion, MassARRAY is a highly sensitive and accurate method for type-specific detection of oncogenic HPV in cervical cancer, with Proofer showing impressive performance.

  13. Dynamics of Theileria orientalis genotype population in cattle in a year-round grazing system.

    PubMed

    Masatani, Tatsunori; Yoshihara, Shunpei; Matsubara, Atsuko; Gotoh, Takafumi; Takahashi, Hideyuki; Tanaka, Tetsuya; Andoh, Masako; Endo, Yasuyuki; Matsuo, Tomohide

    2016-03-01

    Theirelia orientalis is a tick-borne haemoprotozoan parasite, and infection with this parasite is one of the most important diseases for grazing cattle. Co-infection of cattle with different genotypes of T. orientalis often occurs. In this study, we investigated the temporal dynamics of genotypes in cattle in a year-round grazing system in Japan. Genotype-specific PCR assays to determine major piroplasm surface protein (MPSP) genotypes (types 1 to 5) of T. orientalis were performed by using time-course blood samples collected from grazing cattle and ticks in a pasture. All 20 cattle investigated in this study were infected with T. orientalis. By using genotype-specific PCR, we detected the combination of genotypes of T. orientalis (types 1 to 5) from each cattle. These multiple genotypes of T. orientalis were also confirmed in ticks. Notably, each genotype of T. orientalis in cattle was temporally detected from cattle and more variable genotypes were found in summer. The observed temporal dynamics of the MPSP genotypes of T. orientalis in cattle could be explained by host immunity against the parasites or genetic recombination of parasite in ticks. PMID:27078669

  14. High-throughput variation detection and genotyping using microarrays.

    PubMed

    Cutler, D J; Zwick, M E; Carrasquillo, M M; Yohn, C T; Tobin, K P; Kashuk, C; Mathews, D J; Shah, N A; Eichler, E E; Warrington, J A; Chakravarti, A

    2001-11-01

    The genetic dissection of complex traits may ultimately require a large number of SNPs to be genotyped in multiple individuals who exhibit phenotypic variation in a trait of interest. Microarray technology can enable rapid genotyping of variation specific to study samples. To facilitate their use, we have developed an automated statistical method (ABACUS) to analyze microarray hybridization data and applied this method to Affymetrix Variation Detection Arrays (VDAs). ABACUS provides a quality score to individual genotypes, allowing investigators to focus their attention on sites that give accurate information. We have applied ABACUS to an experiment encompassing 32 autosomal and eight X-linked genomic regions, each consisting of approximately 50 kb of unique sequence spanning a 100-kb region, in 40 humans. At sufficiently high-quality scores, we are able to read approximately 80% of all sites. To assess the accuracy of SNP detection, 108 of 108 SNPs have been experimentally confirmed; an additional 371 SNPs have been confirmed electronically. To access the accuracy of diploid genotypes at segregating autosomal sites, we confirmed 1515 of 1515 homozygous calls, and 420 of 423 (99.29%) heterozygotes. In replicate experiments, consisting of independent amplification of identical samples followed by hybridization to distinct microarrays of the same design, genotyping is highly repeatable. In an autosomal replicate experiment, 813,295 of 813,295 genotypes are called identically (including 351 heterozygotes); at an X-linked locus in males (haploid), 841,236 of 841,236 sites are called identically.

  15. Genotype × age interaction in human transcriptional ageing

    PubMed Central

    Kent, Jack W.; Göring, Harald H. H.; Charlesworth, Jac C.; Drigalenko, Eugene; Diego, Vincent P.; Curran, Joanne E.; Johnson, Matthew P.; Dyer, Thomas D.; Cole, Shelley A.; Jowett, Jeremy B. M.; Mahaney, Michael C.; Comuzzie, Anthony G.; Almasy, Laura; Moses, Eric K.; Blangero, John; Williams-Blangero, Sarah

    2012-01-01

    Individual differences in biological ageing (i.e., the rate of physiological response to the passage of time) may be due in part to genotype-specific variation in gene action. However, the sources of heritable variation in human age-related gene expression profiles are largely unknown. We have profiled genome-wide expression in peripheral blood mononuclear cells from 1,240 individuals in large families and found 4,472 human autosomal transcripts, representing ~4,349 genes, significantly correlated with age. We identified 623 transcripts that show genotype by age interaction in addition to a main effect of age, defining a large set of novel candidates for characterization of the mechanisms of differential biological ageing. We applied a novel SNP genotype×age interaction test to one of these candidates, the ubiquilin-like gene UBQLNL, and found evidence of joint cis-association and genotype by age interaction as well as trans-genotype by age interaction for UBQLNL expression. Both UBQLNL expression levels at recruitment and cis genotype are associated with longitudinal cancer risk in our study cohort. PMID:22871458

  16. Comparison of a Baculovirus-Based VP2 Enzyme Immunoassay (EIA) to an Escherichia coli-Based VP1 EIA for Detection of Human Parvovirus B19 Immunoglobulin M and Immunoglobulin G in Sera of Pregnant Women

    PubMed Central

    Jordan, Jeanne A.

    2000-01-01

    A split-sample study was conducted to evaluate the clinical performance of an enzyme immunoassay that detects the human parvovirus B19 virus (B19V) immunoglobulin M (IgM) or IgG in the sera of pregnant women. The initial study compared a baculovirus-expressed VP2 enzyme immunoassay (BVP2 EIA) (Biotrin International Inc., Dublin, Ireland) with the currently available and commonly used Escherichia coli-expressed VP1 enzyme immunoassay (EVP1 EIA) (MRL Diagnostics, Cypress, Calif.). There was a high degree of agreement between the two assays in the detection of IgM antibodies (283 of 307 [92.2%]) or IgG antibodies (279 of 311 [89.7%]), with the majority of discrepancies (IgM, 17 of 24 [71%]; IgG, 16 of 31 [50%]) being due to equivocal data obtained with the EVP1 EIA. Specimens with discordant BVP2 EIA and EVP1 EIA results (23 of 24 IgM and 32 of 32 IgG results) were analyzed further by baculovirus-based VP1 immunofluorescence assays (BVP1 IFAs) (Biotrin International). The BVP2 EIA and BVP1 IFA results for 20 of 23 and 28 of 32 specimens for IgM and IgG, respectively, were concordant. In contrast, the EVP1 EIA and BVP1 IFA data for only 3 of 23 and 4 of 32 specimens for IgM and IgG, respectively, were in agreement, despite the fact that the same capsid antigen was used. Both the BVP2 EIAs and BVP1 IFAs utilize a conformational viral capsid antigen, while the EVP1 EIA uses a denatured viral capsid antigen. In conclusion, the BVP2 EIAs produced far fewer equivocal results for IgM and IgG, correlating more closely to the confirmatory BVP IFAs, than did the EVP1 EIAs and proved to be more accurate for detecting B19V antibodies in the sera of pregnant women. PMID:10747128

  17. Genotype imputation via matrix completion.

    PubMed

    Chi, Eric C; Zhou, Hua; Chen, Gary K; Del Vecchyo, Diego Ortega; Lange, Kenneth

    2013-03-01

    Most current genotype imputation methods are model-based and computationally intensive, taking days to impute one chromosome pair on 1000 people. We describe an efficient genotype imputation method based on matrix completion. Our matrix completion method is implemented in MATLAB and tested on real data from HapMap 3, simulated pedigree data, and simulated low-coverage sequencing data derived from the 1000 Genomes Project. Compared with leading imputation programs, the matrix completion algorithm embodied in our program MENDEL-IMPUTE achieves comparable imputation accuracy while reducing run times significantly. Implementation in a lower-level language such as Fortran or C is apt to further improve computational efficiency. PMID:23233546

  18. Estimation of genotyping error rate from repeat genotyping, unintentional recaptures and known parent-offspring comparisons in 16 microsatellite loci for brown rockfish (Sebastes auriculatus).

    PubMed

    Hess, Maureen A; Rhydderch, James G; LeClair, Larry L; Buckley, Raymond M; Kawase, Mitsuhiro; Hauser, Lorenz

    2012-11-01

    Genotyping errors are present in almost all genetic data and can affect biological conclusions of a study, particularly for studies based on individual identification and parentage. Many statistical approaches can incorporate genotyping errors, but usually need accurate estimates of error rates. Here, we used a new microsatellite data set developed for brown rockfish (Sebastes auriculatus) to estimate genotyping error using three approaches: (i) repeat genotyping 5% of samples, (ii) comparing unintentionally recaptured individuals and (iii) Mendelian inheritance error checking for known parent-offspring pairs. In each data set, we quantified genotyping error rate per allele due to allele drop-out and false alleles. Genotyping error rate per locus revealed an average overall genotyping error rate by direct count of 0.3%, 1.5% and 1.7% (0.002, 0.007 and 0.008 per allele error rate) from replicate genotypes, known parent-offspring pairs and unintentionally recaptured individuals, respectively. By direct-count error estimates, the recapture and known parent-offspring data sets revealed an error rate four times greater than estimated using repeat genotypes. There was no evidence of correlation between error rates and locus variability for all three data sets, and errors appeared to occur randomly over loci in the repeat genotypes, but not in recaptures and parent-offspring comparisons. Furthermore, there was no correlation in locus-specific error rates between any two of the three data sets. Our data suggest that repeat genotyping may underestimate true error rates and may not estimate locus-specific error rates accurately. We therefore suggest using methods for error estimation that correspond to the overall aim of the study (e.g. known parent-offspring comparisons in parentage studies).

  19. Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates.

    PubMed

    Baums, Christoph G; Schotte, Ulrich; Amtsberg, Gunter; Goethe, Ralph

    2004-05-20

    In this study we provide a protocol for genotyping Clostridium perfringens with a new multiplex PCR. This PCR enables reliable and specific detection of the toxin genes cpa, cpb, etx, iap, cpe and cpb2 from heat lysed bacterial suspensions. The efficiency of the protocol was demonstrated by typing C. perfringens reference strains and isolates from veterinary bacteriological routine diagnostic specimens.

  20. Rapid microarray-based genotyping of Chlamydia spp. strains from clinical tissue samples.

    PubMed

    Sachse, Konrad; Ruettger, Anke

    2015-01-01

    Pathogenic Chlamydia (C.) psittaci and C. trachomatis strains can be genotyped based on variations in the ompA genomic locus. In the present chapter, we describe rapid genotyping assays for both chlamydial agents using the ArrayStrip™ (AS) microarray platform. The test is targeting multiple discriminatory sites in the variable domains of the ompA gene by using 35 (C. psittaci) and 61 (C. trachomatis) oligonucleotide probes representing genotype-specific polymorphisms. In addition to discrimination among the established genotypes, this approach allows identification of atypical strains that were not accessible to typing using previously established techniques, such as PCR-RFLP or serotyping. The present DNA microarray assay can be conducted directly on clinical tissue samples and is suitable for tracing epidemiological chains and exploring the dissemination of particular genotypes. The procedure is easy to handle and economically affordable, and it allows genotyping of up to 32 clinical samples per day, thus lending itself for routine diagnosis as well.

  1. Commercially available immunoglobulins contain virus neutralizing antibodies against all major genotypes of polyomavirus BK.

    PubMed

    Randhawa, P; Pastrana, D V; Zeng, G; Huang, Y; Shapiro, R; Sood, P; Puttarajappa, C; Berger, M; Hariharan, S; Buck, C B

    2015-04-01

    Neutralizing antibodies (NAbs) form the basis of immunotherapeutic strategies against many important human viral infections. Accordingly, we studied the prevalence, titer, genotype-specificity, and mechanism of action of anti-polyomavirus BK (BKV) NAbs in commercially available human immune globulin (IG) preparations designed for intravenous (IV) use. Pseudovirions (PsV) of genotypes Ia, Ib2, Ic, II, III, and IV were generated by co-transfecting a reporter plasmid encoding luciferase and expression plasmids containing synthetic codon-modified VP1, VP2, and VP3 capsid protein genes into 293TT cells. NAbs were measured using luminometry. All IG preparations neutralized all BKV genotypes, with mean EC50 titers as high as 254 899 for genotype Ia and 6,666 for genotype IV. Neutralizing titers against genotypes II and III were higher than expected, adding to growing evidence that infections with these genotypes are more common than currently appreciated. Batch to batch variation in different lots of IG was within the limits of experimental error. Antibody mediated virus neutralizing was dose dependent, modestly enhanced by complement, genotype-specific, and achieved without effect on viral aggregation, capsid morphology, elution, or host cell release. IG contains potent NAbs capable of neutralizing all major BKV genotypes. Clinical trials based on sound pharmacokinetic principles are needed to explore prophylactic and therapeutic applications of these anti-viral effects, until effective small molecule inhibitors of BKV replication can be developed.

  2. HCV genotype determination in monoinfected and HIV co-infected patients in Cuba.

    PubMed

    Rodríguez Lay, Licel de Los Angeles; Villalba, Maria Caridad Montalvo; Corredor, Marité Bello; Frómeta, Susel Sariego; Hernández, Jeny Marante; Carrera, Santiago Dueñas; Wong, Meilin Sánchez; Samada, Marcia; Núñez, Milay Bello; Alonso, Lidunka Valdes; da Silva Filho, Hermes Pedreira; Hübschen, Judith M; Reis, Mitermayer G

    2012-12-01

    With the aim to characterize the HCV genotype distribution in Cuba, sera were collected from two subgroups: HCV-monoinfected and HCV/HIV co-infected patients. A combination of reverse transcription-PCR using genotype-specific primers, restriction fragment length polymorphism and sequencing was used to determine the genotype of 84 samples. Seventy-nine (94%) showed single infections (10 [12%] were genotype 1a and 69 [82%] genotype 1b) and 5 (6%) samples corresponded to mixed infections (2 [2%] with genotypes 1a/3a and 1 sample [1%] each with 1b/3a, 1b/4a and 1a/1b/3a). HCV/HIV co-infected subjects had a higher frequency of mixed infections (p=0.08), infection with genotype 3a (p=0.18) and for the first time genotype 4a was found. There was no association of any demographic characteristics with any specific genotype although HCV/HIV co-infected patients showed a tendency to have mixed genotypes in those older than 45 years of age (p=0.11). Phylogenetic analysis showed that HCV isolates clustered with subtypes 1b (n=15, maximal genetic distance 2.51%) and 1a (n=2, maximal genetic distance 0.35%). This report presents the prevalence of HCV genotypes in monoinfected and HIV co-infected patients, mixed HCV infections in HCV/HIV co-infected men who have sex with men with high-risk sexual practices and for the first time identifies that the uncommon genotype 4a can be present in a patient co-infected with HIV.

  3. Prevalence of BPV genotypes in a German cowshed determined by a novel multiplex BPV genotyping assay.

    PubMed

    Schmitt, Markus; Fiedler, Volker; Müller, Martin

    2010-12-01

    Bovine papillomaviruses (BPV) induce benign tumours of the cutaneous or mucosal epithelia in cattle, but are also involved in the development of cancer of the urinary bladder and of the upper gastrointestinal tract. Current BPV genotyping assays employ techniques developed originally for the detection of human papillomaviruses. These methods rely on consensus PCR amplification and subsequent sequencing and are cumbersome and limited in their analytic sensitivity to detect BPV, especially in multiple infections. In this study, a novel multiplex BPV genotyping assay is described to detect sensitively and specifically BPV-1 to -10 as well as BaPV-11. The assay is based on a multiplex PCR using novel broad-spectrum bovine papillomavirus (BSBP) primers followed by multiplex bovine genotyping (MBG) by Luminex xMAP technology. The detection limit of the assay was shown to be between 10 and 100 BPV genomes. In a first application, BPV was detected in 100% of wart preparations with BPV-8 being most prevalent, followed by types 6, 1 and 10. The majority of warts were positive for at least four BPV types. In conclusion, BSBP-PCR/MBG is a powerful high-throughput method suitable for the study of the natural history of BPV and could be useful to veterinarians for the monitoring of the efficacy of future BPV vaccines.

  4. Improved ancestry estimation for both genotyping and sequencing data using projection procrustes analysis and genotype imputation.

    PubMed

    Wang, Chaolong; Zhan, Xiaowei; Liang, Liming; Abecasis, Gonçalo R; Lin, Xihong

    2015-06-01

    Accurate estimation of individual ancestry is important in genetic association studies, especially when a large number of samples are collected from multiple sources. However, existing approaches developed for genome-wide SNP data do not work well with modest amounts of genetic data, such as in targeted sequencing or exome chip genotyping experiments. We propose a statistical framework to estimate individual ancestry in a principal component ancestry map generated by a reference set of individuals. This framework extends and improves upon our previous method for estimating ancestry using low-coverage sequence reads (LASER 1.0) to analyze either genotyping or sequencing data. In particular, we introduce a projection Procrustes analysis approach that uses high-dimensional principal components to estimate ancestry in a low-dimensional reference space. Using extensive simulations and empirical data examples, we show that our new method (LASER 2.0), combined with genotype imputation on the reference individuals, can substantially outperform LASER 1.0 in estimating fine-scale genetic ancestry. Specifically, LASER 2.0 can accurately estimate fine-scale ancestry within Europe using either exome chip genotypes or targeted sequencing data with off-target coverage as low as 0.05×. Under the framework of LASER 2.0, we can estimate individual ancestry in a shared reference space for samples assayed at different loci or by different techniques. Therefore, our ancestry estimation method will accelerate discovery in disease association studies not only by helping model ancestry within individual studies but also by facilitating combined analysis of genetic data from multiple sources. PMID:26027497

  5. [Preliminary study on HLA-B genotyping by oligonucleotide chips].

    PubMed

    Lan, Ke; Hu, Shou-Wang; Zhang, Fan; Wang, Hui; Guan, Wei; Ding, Yu; Sun, Ou-Jun; Wang, Sheng-Qi

    2003-04-01

    HLA genes constitute a highly polymorphic multigene system. In the present study, HLA-B oligonucleotide chips were manufactured by using a set of sequence-specific oligonucleotide probes derived from polymorphic regions in exon 2 and exon 3 of HLA-B gene spotted by microarrayer onto the aldehyde modified glass slides. In addition, the sequenced HLA-B gene clones used as standard samples were amplified from exon 2 and exon 3 by PCR. Together with the correct hybridization and wash conditions, the PCR products were bound with the array probes on the chip, and the hybridization patterns were transformed to HLA-B genotypes. The results showed that the genotypes of standard samples by the HLA-B oligonucleotide chips were completely identical with the sequenced clones. In conclusion, the oligonucleotide chip method presented here for HLA-B genotyping is a rapid, accurate, sensitive and attractive high throughput biochemical way.

  6. Rapid fluorescent lateral-flow immunoassay for hepatitis B virus genotyping.

    PubMed

    Song, Liu-Wei; Wang, Ying-Bin; Fang, Lin-Lin; Wu, Yong; Yang, Lin; Chen, Jie-Yu; Ge, Sheng-Xiang; Zhang, Jing; Xiong, You-Zheng; Deng, Xiu-Mei; Min, Xiao-Ping; Zhang, Jun; Chen, Pei-Jer; Yuan, Quan; Xia, Ning-Shao

    2015-01-01

    Hepatitis B virus (HBV) genotyping plays an important role in the clinical management of chronic hepatitis B (CHB) patients. However, the current nucleic acid based techniques are expensive, time-consuming, and inconvenient. Here, we developed a novel DNA-independent HBV genotyping tool based on a one-step fluorescent lateral flow immunoassay (LFIA). Epitope-targeting immunization and screening techniques were used to develop HBV genotype specific monoclonal antibodies (mAbs). These mAbs were used to develop a multitest LFIA with a matched scanning luminoscope for HBV genotyping (named the GT-LFIA). The performance of this novel assay was carefully evaluated in well-characterized clinical cohorts. The GT-LFIA, which can specifically differentiate HBV genotypes A, B, C, and D in a pretreatment-free single test, was successfully developed using four genotype specific mAbs. The detection limits of the GT-LFIA for HBV genotypes A, B, C, and D were 2.5-10.0 IU HBV surface antigen/mL, respectively. Among the sera from 456 CHB patients, 439 (96.3%; 95% confidence interval (CI), 94.1-97.8%) were genotype-differentiable by the GT-LFIA and 437 (99.5%; 95% CI, 98.4-99.9%) were consistent with viral genome sequencing. In the 21 patients receiving nucleos(t)ide analogue therapy, for end-of-treatment specimens that were HBV DNA undetectable and were not applicable for DNA-dependent genotyping, the GT-LFIA presented genotyping results that were consistent with those obtained in pretreatment specimens by viral genome sequencing and the GT-LFIA. In conclusion, the novel GT-LFIA is a convenient, fast, and reliable tool for differential HBV genotyping, especially in patients with low or undetectable HBV DNA levels. PMID:25892477

  7. Genotype-driven recruitment: a strategy whose time has come?

    PubMed Central

    2013-01-01

    Background Genotype-Driven Recruitment (GDR) is a research design that recruits research participants based on genotype rather than based on the presence or absence of a particular condition or clinical outcome. Analyses of the ethical issues of GDR studies, and the recommendations derived from these analyses, are based on GDR research designs that make use of genetic information already collected in previous studies. However, as genotyping becomes more affordable, it is expected that genotypic information will become a common part of the information stored in biobanks and held in health care records. Furthermore, individuals will increasingly gain knowledge of their own genotypes through Direct-to-Consumer services. One can therefore foresee that individuals will be invited to participate not only in follow-up GDR studies but also in original GDR studies because genetic information about them is available. These individuals may or may have not participated in research before and may or may not be aware that their genetic information is available for research. Discussion From a conceptual point of view, we investigate whether the current ethics-related recommendations for the conduct of GDR suffice for a broader array of circumstances under which genetic information can be available. Our analysis reveals that the existing recommendations do not suffice for a broader use of GDR. Summary Our findings refocus attention on ethical issues which are neither new nor specific to GDR but which place greater demand on coordinated solutions. These challenges and approaches for addressing them are discussed. PMID:23702358

  8. Phylogenetic Analysis of Hepatitis B Virus Genotypes Circulating in Different Risk Groups of Panama, Evidence of the Introduction of Genotype A2 in the Country

    PubMed Central

    Martínez, Alexander A.; Zaldívar, Yamitzel; Arteaga, Griselda; de Castillo, Zoila; Ortiz, Alma; Mendoza, Yaxelis; Castillero, Omar; Castillo, Juan A.; Cristina, Juan; Pascale, Juan M.

    2015-01-01

    The Hepatitis B Virus (HBV) can cause acute or chronic infection it is also associated with the development of liver cancer, thousands of new infections occur on a yearly basis, and many of these cases are located in certain areas of the Caribbean and Latin America. In these areas, the HBV prevalence is still high which makes this virus a serious public health concern to the entire region. Studies performed in Panama suggest a complex pattern in the distribution of HBV among the country’s different risk groups. We use phylogenetic analysis in order to determine which HBV genotypes were circulating in these specific groups; for this we used a fragment of the PreS2/2 region of the HBV genome. Subsequently whole HBV genome sequences were used for Bayesian analysis of phylodynamics and phylogeography. Two main genotypes were found: genotype A (54.5%) and genotype F (45.5%). There was a difference in the distribution of genotypes according to risk groups: 72.9% of high risk groups were associated to genotype A, and 55.0% of samples of genotype F were associated to the low risk group (p<0.002). The Bayesian analysis of phylogeny-traits association revealed a statistically significant geographical association (p<0.0001) with both genotypes and different regions of the country. The Bayesian time of most recent common ancestor analysis (tMRCA) revealed a recent tMRCA for genotype A2 circulating in Panama (1997, 95% HPD: 1986—2005), when it is compared with Panamanian genotype F1c sequences (1930, 95% HPD: 1810 – 2005). These results suggest a possible change in the distribution of HBV genotypes in Panama and Latin America as a whole. They also serve to encourage the implementation of vaccination programs in high-risk groups, in order to prevent an increase in the number of new HBV cases in Latin America and worldwide. PMID:26230260

  9. Mycobacterium tuberculosis Beijing genotype, northern Malawi.

    PubMed

    Glynn, Judith R; Crampin, Amelia C; Traore, Hamidou; Yates, Malcolm D; Mwaungulu, Frank D; Ngwira, Bagrey M; Chaguluka, Steven D; Mwafulirwa, Donex T; Floyd, Sian; Murphy, Caroline; Drobniewski, Francis A; Fine, Paul E M

    2005-01-01

    In a 7-year population-based study in Malawi, we showed that Beijing genotype tuberculosis (TB) increased as a proportion of TB cases. All the Beijing genotype strains were fully drug sensitive. Contact histories, TB type, and case-fatality rates were similar for Beijing and non-Beijing genotype TB.

  10. Whole-canopy carbon gain as a result of selection on individual performance of ten genotypes of a clonal plant.

    PubMed

    Vermeulen, Peter J; Anten, Niels P R; Stuefer, Josef F; During, Heinjo J

    2013-06-01

    Game theoretical models predict that plant competition for light leads to reduced productivity of vegetation stands through selection for traits that maximize carbon gains of individuals. Using empirical results from a 5-year competition experiment with 10 genotypes of the clonal plant Potentilla reptans, we tested this prediction by analyzing the effects of the existing leaf area values on the carbon gain of the different genotypes and the consequent whole canopy carbon gain. We focused on specific leaf area (SLA) due to its role in the trade-off between light capture area and photosynthetic capacity per unit area. By combining a canopy model based on measured leaf area and light profiles with a game theoretical approach, we analyzed how changes in the SLA affected genotypic and whole-stand carbon gain. This showed that all genotypes contributed to reduced stand productivity. The dominant genotype maximized its share of total carbon gain, resulting in lower than maximal absolute gain. Other genotypes did not maximize their share. Hypothetical mutants of the dominant genotype were not able to achieve a higher carbon gain. Conversely, in other genotypes, some mutations did result in increased carbon gain. Hence, genotypic differences in the ability to maximize performance may determine genotype frequency. It shows how genotypic selection may result in lower carbon gains of the whole vegetation, and of the individual genotypes it consists of, through similar mechanisms as those that lead to the tragedy of the commons. PMID:23114427

  11. Selection of common bean (Phaseolus vulgaris L.) genotypes using a genotype plus genotype x environment interaction biplot.

    PubMed

    Corrêa, A M; Teodoro, P E; Gonçalves, M C; Santos, A; Torres, F E

    2016-01-01

    Recently, the genotype plus genotype x environment interaction (GGE) biplot methodology has been used to investigate genotype x environment interactions in several crop species, but has not been applied to the common bean (Phaseolus vulgaris L.) crop in Brazil. The aim of this study was to identify common bean genotypes that exhibit high grain yield and stability in the State of Mato Grosso do Sul, Brazil. We conducted 12 trials from 2000 to 2006 in the municipalities of Aquidauana and Dourados, and evaluated 13 genotypes in a randomized block design with three replications. Grain yield data were subjected to individual and joint analyses of variance. After analyzing the GE interaction, the adaptability and phenotypic stability of the common bean genotypes were analyzed using GGE biplot methodology. The genotypes EMGOPA-201, Xamego, and Aporé are recommended for growing in Mato Grosso do Sul, because they exhibited high grain yield and phenotypic stability. PMID:27525915

  12. Echinococcus granulosus sensu lato genotypes infecting humans--review of current knowledge.

    PubMed

    Alvarez Rojas, Cristian A; Romig, Thomas; Lightowlers, Marshall W

    2014-01-01

    Genetic variability in the species group Echinococcus granulosus sensu lato is well recognised as affecting intermediate host susceptibility and other biological features of the parasites. Molecular methods have allowed discrimination of different genotypes (G1-10 and the 'lion strain'), some of which are now considered separate species. An accumulation of genotypic analyses undertaken on parasite isolates from human cases of cystic echinococcosis provides the basis upon which an assessment is made here of the relative contribution of the different genotypes to human disease. The allocation of samples to G-numbers becomes increasingly difficult, because much more variability than previously recognised exists in the genotypic clusters G1-3 (=E. granulosus sensu stricto) and G6-10 (Echinococcus canadensis). To accommodate the heterogeneous criteria used for genotyping in the literature, we restrict ourselves to differentiate between E. granulosus sensu stricto (G1-3), Echinococcus equinus (G4), Echinococcus ortleppi (G5) and E. canadensis (G6-7, G8, G10). The genotype G1 is responsible for the great majority of human cystic echinococcosis worldwide (88.44%), has the most cosmopolitan distribution and is often associated with transmission via sheep as intermediate hosts. The closely related genotypes G6 and G7 cause a significant number of human infections (11.07%). The genotype G6 was found to be responsible for 7.34% of infections worldwide. This strain is known from Africa and Asia, where it is transmitted mainly by camels (and goats), and South America, where it appears to be mainly transmitted by goats. The G7 genotype has been responsible for 3.73% of human cases of cystic echinococcosis in eastern European countries, where the parasite is transmitted by pigs. Some of the samples (11) could not be identified with a single specific genotype belonging to E. canadensis (G6/10). Rare cases of human cystic echinococcosis have been identified as having been caused by

  13. Glutamine synthetase in Durum Wheat: Genotypic Variation and Relationship with Grain Protein Content.

    PubMed

    Nigro, Domenica; Fortunato, Stefania; Giove, Stefania L; Paradiso, Annalisa; Gu, Yong Q; Blanco, Antonio; de Pinto, Maria C; Gadaleta, Agata

    2016-01-01

    Grain protein content (GPC), is one of the most important trait in wheat and its characterized by a very complex genetic control. The identification of wheat varieties with high GPC (HGPC), as well as the characterization of central enzymes involved in these processes, are important for more sustainable agricultural practices. In this study, we focused on Glutamine synthetase (GS) as a candidate to study GPC in wheat. We analyzed GS expression and its enzymatic activity in different tissues and phenological stages in 10 durum wheat genotypes with different GPC. Although each genotype performed quite differently from the others, both because their genetic variability and their adaptability to specific environmental conditions, the highest GS activity and expression were found in genotypes with HGPC and vice versa the lowest ones in genotypes with low GPC (LGPC). Moreover, in genotypes contrasting in GPC bred at different nitrogen regimes (0, 60, 140 N Unit/ha) GS behaved differently in diverse organs. Nitrogen supplement increased GS expression and activity in roots of all genotypes, highlighting the key role of this enzyme in nitrogen assimilation and ammonium detoxification in roots. Otherwise, nitrogen treatments decreased GS expression and activity in the leaves of HGPC genotypes and did not affect GS in the leaves of LGPC genotypes. Finally, no changes in GS and soluble protein content occurred at the filling stage in the caryopses of all analyzed genotypes.

  14. Glutamine synthetase in Durum Wheat: Genotypic Variation and Relationship with Grain Protein Content.

    PubMed

    Nigro, Domenica; Fortunato, Stefania; Giove, Stefania L; Paradiso, Annalisa; Gu, Yong Q; Blanco, Antonio; de Pinto, Maria C; Gadaleta, Agata

    2016-01-01

    Grain protein content (GPC), is one of the most important trait in wheat and its characterized by a very complex genetic control. The identification of wheat varieties with high GPC (HGPC), as well as the characterization of central enzymes involved in these processes, are important for more sustainable agricultural practices. In this study, we focused on Glutamine synthetase (GS) as a candidate to study GPC in wheat. We analyzed GS expression and its enzymatic activity in different tissues and phenological stages in 10 durum wheat genotypes with different GPC. Although each genotype performed quite differently from the others, both because their genetic variability and their adaptability to specific environmental conditions, the highest GS activity and expression were found in genotypes with HGPC and vice versa the lowest ones in genotypes with low GPC (LGPC). Moreover, in genotypes contrasting in GPC bred at different nitrogen regimes (0, 60, 140 N Unit/ha) GS behaved differently in diverse organs. Nitrogen supplement increased GS expression and activity in roots of all genotypes, highlighting the key role of this enzyme in nitrogen assimilation and ammonium detoxification in roots. Otherwise, nitrogen treatments decreased GS expression and activity in the leaves of HGPC genotypes and did not affect GS in the leaves of LGPC genotypes. Finally, no changes in GS and soluble protein content occurred at the filling stage in the caryopses of all analyzed genotypes. PMID:27468287

  15. Glutamine synthetase in Durum Wheat: Genotypic Variation and Relationship with Grain Protein Content

    PubMed Central

    Nigro, Domenica; Fortunato, Stefania; Giove, Stefania L.; Paradiso, Annalisa; Gu, Yong Q.; Blanco, Antonio; de Pinto, Maria C.; Gadaleta, Agata

    2016-01-01

    Grain protein content (GPC), is one of the most important trait in wheat and its characterized by a very complex genetic control. The identification of wheat varieties with high GPC (HGPC), as well as the characterization of central enzymes involved in these processes, are important for more sustainable agricultural practices. In this study, we focused on Glutamine synthetase (GS) as a candidate to study GPC in wheat. We analyzed GS expression and its enzymatic activity in different tissues and phenological stages in 10 durum wheat genotypes with different GPC. Although each genotype performed quite differently from the others, both because their genetic variability and their adaptability to specific environmental conditions, the highest GS activity and expression were found in genotypes with HGPC and vice versa the lowest ones in genotypes with low GPC (LGPC). Moreover, in genotypes contrasting in GPC bred at different nitrogen regimes (0, 60, 140 N Unit/ha) GS behaved differently in diverse organs. Nitrogen supplement increased GS expression and activity in roots of all genotypes, highlighting the key role of this enzyme in nitrogen assimilation and ammonium detoxification in roots. Otherwise, nitrogen treatments decreased GS expression and activity in the leaves of HGPC genotypes and did not affect GS in the leaves of LGPC genotypes. Finally, no changes in GS and soluble protein content occurred at the filling stage in the caryopses of all analyzed genotypes. PMID:27468287

  16. Genotyping and molecular analysis of Enterocytozoon bieneusi isolated from immunocompromised patients in Iran.

    PubMed

    Mirjalali, Hamed; Mirhendi, Hossein; Meamar, Ahmad Reza; Mohebali, Mehdi; Askari, Zeynab; Mirsamadi, Elnaz Sadat; Rezaeian, Mostafa

    2015-12-01

    Microsporidia are known as opportunistic unicellular pathogens, particularly so in individuals with congenital or acquired immunodeficiency. Enterocytozoon bieneusi is one of the most common species infecting both immunocompromised and immunocompetent individuals. The aim of this study was to assess the distribution of E. bieneusi genotypes among immunocompromised patients in Iran. From 329 stool samples referred for parasitological analysis during 2011-2014, 14 samples from immunocompromised patients proving positive for E. bieneusi by SSU rDNA analysis were selected. Genotyping was carried out using specific primers targeting the Internal Transcribed Spacer (ITS) region. Subsequently, all samples were sequenced and results queried against the GenBank database. Moreover, sequences were subject to phylogenetic analysis. The expected amplification product was generated for all samples. Genotype D was identified in patients with HIV+/AIDS, transplant recipients, and cancer patients, while Genotype E was identified only in cancer and HIV+/AIDS patients. Phylogenetic analysis revealed that there was no relationship between genotypes and types of immunosuppression, whereas most genotype D isolates grouped with those described previously from cattle, horses, birds, and humans. E. bieneusi genotype D appears to be the most frequent genotype in immunocompromised patients, while Genotype E was observed only in HIV+/AIDS patients and cancer patients, not transplant recipients.

  17. Technologies for individual genotyping: detection of genetic polymorphisms in drug targets and disease genes.

    PubMed

    Shi, Michael M

    2002-01-01

    Genetic variations have been associated with a predisposition to common diseases and individual variations in drug responses. Identification and genotyping a vast number of genetic polymorphisms in large populations are increasingly important for disease gene identification and pharmacogenetics. Commonly used gel electrophoresis-based genotyping methods for known polymorphisms include polymerase chain reaction (PCR) coupled with restriction fragment-length polymorphism analysis, allele-specific amplification, and oligonucleotide ligation assay. Fluorescent dye-based DNA fragmentation has been extensively used for high-throughput microsatellite or short tandem-repeat genotyping. TaqMan and molecular beacon genotyping are commonly used homogeneous solution hybridization technologies. Because of the ease of experimental assay design, single nucleotide polymorphism (SNP) genotyping methods based on single-base extension are in rapid development, such as fluorescence homogenous assays, pyrosequencing and mass spectrometry. Non-PCR based genotyping assays such as Invader trade mark assays are promised to genotype directly from genomic DNA without the requirement of PCR amplification. The DNA microarray is a solid phase genotyping format that is rapidly developing for parallel genotyping of a large number of SNPs simultaneously. Advanced technologies to identify genetic polymorphisms rapidly, accurately, and cost effectively will fundamentally change the practice of medicine by allowing physicians to prescribe medicine based on a patient's genetic make-up.

  18. Mineral composition of organically grown wheat genotypes: contribution to daily minerals intake.

    PubMed

    Hussain, Abrar; Larsson, Hans; Kuktaite, Ramune; Johansson, Eva

    2010-09-01

    In this study, 321 winter and spring wheat genotypes were analysed for twelve nutritionally important minerals (B, Cu, Fe, Se, Mg, Zn, Ca, Mn, Mo, P, S and K). Some of the genotypes used were from multiple locations and years, resulting in a total number of 493 samples. Investigated genotypes were divided into six genotype groups i.e., selections, old landraces, primitive wheat, spelt, old cultivars and cultivars. For some of the investigated minerals higher concentrations were observed in selections, primitive wheat, and old cultivars as compared to more modern wheat material, e.g., cultivars and spelt wheat. Location was found to have a significant effect on mineral concentration for all genotype groups, although for primitive wheat, genotype had a higher impact than location. Spring wheat was observed to have significantly higher values for B, Cu, Fe, Zn, Ca, S and K as compared to winter wheat. Higher levels of several minerals were observed in the present study, as compared to previous studies carried out in inorganic systems, indicating that organic conditions with suitable genotypes may enhance mineral concentration in wheat grain. This study also showed that a very high mineral concentration, close to daily requirements, can be produced by growing specific primitive wheat genotypes in an organic farming system. Thus, by selecting genotypes for further breeding, nutritional value of the wheat flour for human consumption can be improved.

  19. Estimating genotypes with independently sampled descent graphs.

    PubMed

    Henshall, J M; Tier, B; Kerr, R J

    2001-12-01

    A method for estimating genotypic and identity-by-descent probabilities in complex pedigrees is described. The method consists of an algorithm for drawing independent genotype samples which are consistent with the pedigree and observed genotype. The probability distribution function for samples obtained using the algorithm can be evaluated up to a normalizing constant, and combined with the likelihood to produce a weight for each sample. Importance sampling is then used to estimate genotypic and identity-by-descent probabilities. On small but complex pedigrees, the genotypic probability estimates are demonstrated to be empirically unbiased. On large complex pedigrees, while the algorithm for obtaining genotype samples is feasible, importance sampling may require an infeasible number of samples to estimate genotypic probabilities with accuracy.

  20. The E1B19K-deleted oncolytic adenovirus mutant AdΔ19K sensitizes pancreatic cancer cells to drug-induced DNA-damage by down-regulating Claspin and Mre11

    PubMed Central

    Pantelidou, Constantia; Cherubini, Gioia; Lemoine, Nick R.; Halldén, Gunnel

    2016-01-01

    Adenovirus-mediated sensitization of cancer cells to cytotoxic drugs depends on simultaneous interactions of early viral genes with cell death and survival pathways. It is unclear what cellular factors mediate these interactions in the presence of DNA-damaging drugs. We found that adenovirus prevents Chk1-mediated checkpoint activation through inactivation of Mre11 and downregulation of the pChk1 adaptor-protein, Claspin, in cells with high levels of DNA-damage induced by the cytotoxic drugs gemcitabine and irinotecan. The mechanisms for Claspin downregulation involve decreased transcription and increased degradation, further attenuating pChk1-mediated signalling. Live cell imaging demonstrated that low doses of gemcitabine caused multiple mitotic aberrations including multipolar spindles, micro- and multi-nucleation and cytokinesis failure. A mutant virus with the anti-apoptotic E1B19K-gene deleted (AdΔ19K) further enhanced cell killing, Claspin downregulation, and potentiated drug-induced DNA damage and mitotic aberrations. Decreased Claspin expression and inactivation of Mre11 contributed to the enhanced cell killing in combination with DNA-damaging drugs. These results reveal novel mechanisms that are utilised by adenovirus to ensure completion of its life cycle in the presence of cellular DNA damage. Taken together, our findings reveal novel cellular targets that may be exploited when developing improved anti-cancer therapeutics. PMID:26872382

  1. A Rapid Genotyping Assay for Segregating Human Olfactory Receptor Pseudogenes

    PubMed Central

    Hinkley, Craig S.; Ismaili, Lindita

    2012-01-01

    Variation in odor perception between individuals is initiated by binding of “odorant” molecules to olfactory receptors (ORs) located in the nasal cavity. To determine the mechanism for variation in odor perception, identification of specific ligands for a large number of ORs is required. However, it has been difficult to identify specific ligands, and ligands have been identified for only 2–3% of the hundreds of mammalian ORs. One way to increase the number of identified ligands is to take advantage of >60 human OR genes that are segregating as a result of a single nucleotide polymorphism, between a functional intact allele and a nonfunctional pseudogene allele. Potential ligands for these ORs can be identified by correlating odor perception of an individual with their genotype [intact/intact (I/I) vs. pseudogene/pseudogene (P/P)] for an OR gene. For this type of study, genotypes must be determined for a large number of individuals. We have developed a PCR-based assay to distinguish between the intact and pseudogene alleles of 49 segregating human OR genes and to determine an individual's genotype for these genes. To facilitate rapid determination of genotypes for a large number of individuals, the assay uses a small number of simple steps and equipment commonly found in most molecular biology and biochemistry laboratories. Although this assay was developed to distinguish between polymorphisms in OR genes, it can easily be adapted for use in distinguishing single nucleotide polymorphisms in any gene or chromosomal locus. PMID:23002384

  2. Genotyping NAT2 with only two SNPs (rs1041983 and rs1801280) outperforms the tagging SNP rs1495741 and is equivalent to the conventional 7-SNP NAT2 genotype.

    PubMed

    Selinski, Silvia; Blaszkewicz, Meinolf; Lehmann, Marie-Louise; Ovsiannikov, Daniel; Moormann, Oliver; Guballa, Christoph; Kress, Alexander; Truss, Michael C; Gerullis, Holger; Otto, Thomas; Barski, Dimitri; Niegisch, Günter; Albers, Peter; Frees, Sebastian; Brenner, Walburgis; Thüroff, Joachim W; Angeli-Greaves, Miriam; Seidel, Thilo; Roth, Gerhard; Dietrich, Holger; Ebbinghaus, Rainer; Prager, Hans M; Bolt, Hermann M; Falkenstein, Michael; Zimmermann, Anna; Klein, Torsten; Reckwitz, Thomas; Roemer, Hermann C; Löhlein, Dietrich; Weistenhöfer, Wobbeke; Schöps, Wolfgang; Hassan Rizvi, Syed Adibul; Aslam, Muhammad; Bánfi, Gergely; Romics, Imre; Steffens, Michael; Ekici, Arif B; Winterpacht, Andreas; Ickstadt, Katja; Schwender, Holger; Hengstler, Jan G; Golka, Klaus

    2011-10-01

    Genotyping N-acetyltransferase 2 (NAT2) is of high relevance for individualized dosing of antituberculosis drugs and bladder cancer epidemiology. In this study we compared a recently published tagging single nucleotide polymorphism (SNP) (rs1495741) to the conventional 7-SNP genotype (G191A, C282T, T341C, C481T, G590A, A803G and G857A haplotype pairs) and systematically analysed if novel SNP combinations outperform the latter. For this purpose, we studied 3177 individuals by PCR and phenotyped 344 individuals by the caffeine test. Although the tagSNP and the 7-SNP genotype showed a high degree of correlation (R=0.933, P<0.0001) the 7-SNP genotype nevertheless outperformed the tagging SNP with respect to specificity (1.0 vs. 0.9444, P=0.0065). Considering all possible SNP combinations in a receiver operating characteristic analysis we identified a 2-SNP genotype (C282T, T341C) that outperformed the tagging SNP and was equivalent to the 7-SNP genotype. The 2-SNP genotype predicted the correct phenotype with a sensitivity of 0.8643 and a specificity of 1.0. In addition, it predicted the 7-SNP genotype with sensitivity and specificity of 0.9993 and 0.9880, respectively. The prediction of the NAT2 genotype by the 2-SNP genotype performed similar in populations of Caucasian, Venezuelan and Pakistani background. A 2-SNP genotype predicts NAT2 phenotypes with similar sensitivity and specificity as the conventional 7-SNP genotype. This procedure represents a facilitation in individualized dosing of NAT2 substrates without losing sensitivity or specificity.

  3. [Root morphological characteristics of barley genotype with high phosphorus efficiency under phosphorus stress].

    PubMed

    Chen, Hai-ying; Yu, Hai-ying; Chen, Guang-deng; Li, Ting-xuan

    2015-10-01

    A pot experiment was carried out to test the effects of phosphorus (P) supply levels (25, 50, and 75 mg P2O5 . kg-1) with two P genotype (efficient DH110+ and DH147, inefficient DH49) barleys on root morphology and the relationships between root morphology and P uptake. The results showed that barley biomass and P uptake were significantly reduced by low P stress. Efficient genotype barley biomass and P uptake were 1.24-1.70 and 1.18-1.83 times as much as those of inefficient genotype barley respectively. The total root length, total root surface area, average root diameter, adventitious root length and root surface area, lateral root length and root surface area of P efficient genotype barley were significantly reduced with decreasing the P supply level in soil. The total root length, total root surface area, specific root length, lateral root length and surface area of P efficient genotype barley were 1.46-2.06, 1.12-1.51, 1.35-1.72, 1.69-2.42; and 1.40-1.78 times as much as that of those of P inefficient genotype barley, respectively, while the average root diameter was 70.6% - 90.2% of P inefficient genotype barley. Principal component analysis showed that the average root diameter, specific root surface area and specific root length could be used to distinguish two P genotype barleys. Partial least squares regression analysis showed that the total root length, total root surface area made great contributions to P uptake of barley in soil. The contribution of the adventitious root length and surface area on P uptake of barley decreased significantly and the average root diameter, specific root length, lateral root length and root surface area increased with the decreasing P supply level in soil. P efficient genotype barley adapted to low P stress through maintaining the lateral root growth, increasing the specific root length and root fineness. PMID:26995909

  4. [Root morphological characteristics of barley genotype with high phosphorus efficiency under phosphorus stress].

    PubMed

    Chen, Hai-ying; Yu, Hai-ying; Chen, Guang-deng; Li, Ting-xuan

    2015-10-01

    A pot experiment was carried out to test the effects of phosphorus (P) supply levels (25, 50, and 75 mg P2O5 . kg-1) with two P genotype (efficient DH110+ and DH147, inefficient DH49) barleys on root morphology and the relationships between root morphology and P uptake. The results showed that barley biomass and P uptake were significantly reduced by low P stress. Efficient genotype barley biomass and P uptake were 1.24-1.70 and 1.18-1.83 times as much as those of inefficient genotype barley respectively. The total root length, total root surface area, average root diameter, adventitious root length and root surface area, lateral root length and root surface area of P efficient genotype barley were significantly reduced with decreasing the P supply level in soil. The total root length, total root surface area, specific root length, lateral root length and surface area of P efficient genotype barley were 1.46-2.06, 1.12-1.51, 1.35-1.72, 1.69-2.42; and 1.40-1.78 times as much as that of those of P inefficient genotype barley, respectively, while the average root diameter was 70.6% - 90.2% of P inefficient genotype barley. Principal component analysis showed that the average root diameter, specific root surface area and specific root length could be used to distinguish two P genotype barleys. Partial least squares regression analysis showed that the total root length, total root surface area made great contributions to P uptake of barley in soil. The contribution of the adventitious root length and surface area on P uptake of barley decreased significantly and the average root diameter, specific root length, lateral root length and root surface area increased with the decreasing P supply level in soil. P efficient genotype barley adapted to low P stress through maintaining the lateral root growth, increasing the specific root length and root fineness.

  5. New hepatitis C virus (HCV) genotyping system that allows for identification of HCV genotypes 1a, 1b, 2a, 2b, 3a, 3b, 4, 5a, and 6a.

    PubMed Central

    Ohno, O; Mizokami, M; Wu, R R; Saleh, M G; Ohba, K; Orito, E; Mukaide, M; Williams, R; Lau, J Y

    1997-01-01

    Recent studies have focused on whether different hepatitis C virus (HCV) genotypes are associated with different profiles of pathogenicity, infectivity, and response to antiviral therapy. The establishment of a simple and precise genotyping system for HCV is essential to address these issues. A new genotyping system based on PCR of the core region with genotype-specific PCR primers for the determination of HCV genotypes 1a, 1b, 2a, 2b, 3a, 3b, 4, 5a, and 6a was developed. A total of 607 samples (379 from Japan, 63 from the United States, 53 from Korea, 35 from Taiwan, 32 from China, 20 from Hong Kong, 15 from Australia, 6 from Egypt, 3 from Bangladesh, and 1 from South Africa) were tested by both the assay of Okamoto et al. (H. Okamoto, Y. Sugiyama, S. Okada, K. Kurai, Y. Akahane, Y. Sugai, T. Tanaka, K. Sato, F. Tsuda, Y. Miyamura, and M. Mayumi, J. Gen. Virol. 73:673-679, 1992) and this new genotyping system. Comparison of the results showed concordant results for 539 samples (88.8%). Of the 68 samples with discordant results, the nucleotide sequences of the HCV isolates were determined in 23, and their genotypes were determined by molecular evolutionary analysis. In all 23 samples, the assignment of genotype by our new genotyping system was correct. This genotyping system may be useful for large-scale determination of HCV genotypes in clinical studies. PMID:8968908

  6. Immunogenicity difference between two hepatitis E vaccines derived from genotype 1 and 4.

    PubMed

    Wen, Jiyue; Behloul, Nouredine; Dai, Xing; Dong, Chen; Liang, Jiuhong; Zhang, Min; Shi, Chengbo; Meng, Jihong

    2016-04-01

    We investigated the immunogenicity difference between hepatitis E vaccine p239 derived from hepatitis E virus (HEV) genotype 1 and vaccine p179 derived from HEV genotype 4; and the presence of genotype-specific neutralizing epitopes. HEV ORF2 recombinant proteins (p166W01, p166Mex, p166US and p166Chn) derived from the four HEV genotypes were used to detect anti-HEV IgGs in sera of mice and humans vaccinated with p179 or p239 and in sera of rhesus monkey challenged with HEV genotype 1 or 4 strains. Then monoclonal antibodies (mAbs) against genotype 1 or 4 ORF2 recombinant proteins were prepared and their immunoreactivity was assessed using ELISA and Western blotting; their neutralizing activity was evaluated by an in vitro PCR-based neutralization assay. The results revealed significant immunogenicity difference between the two vaccines: p239-induced IgGs reacted more strongly against p166W01 and p166Mex than against p166US and p166Chn in mice and humans. By contrast, p179-induced IgGs showed a stronger reactivity against p166US and p166Chn than against p166W01 and p166Mex. This difference has also been observed in the sera of rhesus monkeys challenged with HEV genotype 1 or 4 strains. Moreover, besides the two common neutralizing mAbs 3G1 and 5G5, two genotype-specific neutralizing mAbs, 2B1 and 4C5, were obtained. 2B1 could specifically bind to recombinant proteins derived from genotypes 1 and 2 and neutralized only genotypes 1 and 2 strains, while 4C5 immunoreacted specifically against recombinant proteins derived from genotypes 3 and 4 and neutralized only genotypes 3 and 4 strains. These findings revealed the existence of immunogenicity difference between the p179 and p239 vaccines and demonstrated that this difference could be due to the presence of HEV genotype-specific neutralization epitopes.

  7. Genotypic variation in a foundation tree (Populus tremula L.) explains community structure of associated epiphytes

    PubMed Central

    Davies, Chantel; Ellis, Christopher J.; Iason, Glenn R.; Ennos, Richard A.

    2014-01-01

    Community genetics hypothesizes that within a foundation species, the genotype of an individual significantly influences the assemblage of dependent organisms. To assess whether these intra-specific genetic effects are ecologically important, it is required to compare their impact on dependent organisms with that attributable to environmental variation experienced over relevant spatial scales. We assessed bark epiphytes on 27 aspen (Populus tremula L.) genotypes grown in a randomized experimental array at two contrasting sites spanning the environmental conditions from which the aspen genotypes were collected. We found that variation in aspen genotype significantly influenced bark epiphyte community composition, and to the same degree as environmental variation between the test sites. We conclude that maintaining genotypic diversity of foundation species may be crucial for conservation of associated biodiversity. PMID:24789141

  8. Six Novel O Genotypes from Shiga Toxin-Producing Escherichia coli.

    PubMed

    Iguchi, Atsushi; Iyoda, Sunao; Seto, Kazuko; Nishii, Hironobu; Ohnishi, Makoto; Mekata, Hirohisa; Ogura, Yoshitoshi; Hayashi, Tetsuya

    2016-01-01

    Serotyping is one of the typing techniques used to classify strains within the same species. O-serogroup diversification shows a strong association with the genetic diversity of O-antigen biosynthesis genes. In a previous study, based on the O-antigen biosynthesis gene cluster (O-AGC) sequences of 184 known Escherichia coli O serogroups (from O1 to O187), we developed a comprehensive and practical molecular O serogrouping (O genotyping) platform using a polymerase chain reaction (PCR) method, named E. coli O-genotyping PCR. Although, the validation assay using the PCR system showed that most of the tested strains were successfully classified into one of the O genotypes, it was impossible to classify 6.1% (35/575) of the strains, suggesting the presence of novel O genotypes. In this study, we conducted sequence analysis of O-AGCs from O-genotype untypeable Shiga toxin-producing E. coli (STEC) strains and identified six novel O genotypes; OgN1, OgN8, OgN9, OgN10, OgN12 and OgN31, with unique wzx and/or wzy O-antigen processing gene sequences. Additionally, to identify these novel O-genotypes, we designed specific PCR primers. A screen of O genotypes using O-genotype untypeable strains showed 13 STEC strains were classified into five novel O genotypes. The O genotyping at the molecular level of the O-AGC would aid in the characterization of E. coli isolates and will assist future studies in STEC epidemiology and phylogeny.

  9. Distribution of Hepatitis B virus genotypes among healthy blood donors in eastern part of North India

    PubMed Central

    Kumar, Kailash; Kumar, Manoj; Rahaman, Sk. H.; Singh, T. B.; Patel, Saurabh Kumar; Nath, Gopal

    2011-01-01

    Aim: We evaluated the distribution HBV genotypes among non-remunerated healthy blood donors in eastern North India. Materials and Methods: During screening of donated blood, 176 consecutive HBsAg positive, samples comprised the study. HBV-DNA was quantitative detected in 150 samples by PCR. HBV genotype was determined by identifying genotype-specific DNA band using nested PCR. Results: Majorities were of age group 31-40 yrs (65.3%). Males (92.7%) outnumbered females (7.3%) and were HbeAg-negative HBsAg carriers. Over all, genotype-A was the most prevalent (54%) followed by D (21.3%). We did not find genotype-G and H. Districts under study, divided into four zones: Zone–I genotype-A was most common (62.3%) followed by D (18.8%); Zone–II genotype–C (41.2%) was more frequent followed by D (20.6% and A (17.7%). Zone–III in adjoining Bihar state close to Zone–I, A was more prevalent (81.8%) followed by B and C (9.1%). In Zone-IV adjoining Zone- II had genotype-A (100%) only. Genotype–D had more sporadic distribution. Genotype-E and F were prevalent in Zone I and II (3/150, 2%). Conclusions: Among blood donors HBV genotype-A followed by D was the most prevalent in eastern North India. Genotype–A had pattern of distribution signifying common focus, while D was more sporadic and C had single large pocket (Zone-II) probably common focus but restricting to particular area. Evidences are suggestive of association of HBV genotype in liver dysfunction. An effective treatment and preventive strategies based of genotypes will reduce the disease burden and increase the blood safety. PMID:21897593

  10. Characterization of the eg95 gene family in the G6 genotype of Echinococcus granulosus.

    PubMed

    Alvarez Rojas, Cristian A; Gauci, Charles G; Nolan, Matthew J; Harandi, Majid Fasihi; Lightowlers, Marshall W

    2012-06-01

    Cystic echinococcosis in humans and livestock animals is caused by infection with the cestode parasite Echinococcus granulosus. A number of genotypes of the parasite (designated G1-G10) are known to exist, with the genotype cluster G1-G3 and genotype G6 being responsible for the majority of humans infections. A recombinant vaccine has been developed for use in livestock to prevent infection with E. granulosus. The vaccine is based on the antigen EG95 which is expressed in the early larval stage (oncosphere) of the parasite. The EG95 antigen was originally cloned from the G1 genotype of E. granulosus and the protein has been found to be encoded by members of a small family of related genes in this genotype. Reliable information has not been available about the likely efficacy of the EG95 vaccine against genotypes other than G1. In this study, genomic DNA cloning techniques were used to characterize seven eg95-related gene fragments from the G6 genotype of E. granulosus. Three proteins appear to be encoded by these genes. Considerable differences were found between the EG95 related proteins from the G6 genotype compared with the EG95 protein from the G1 genotype. These differences suggest that the EG95-related proteins from the G6 genotype may have different antigenic epitopes compared with the current vaccine antigen. Data presented in this study have implications for future vaccine design and provide the information that would enable a G6 genotype-specific vaccine to be developed against E. granulosus, should this be considered a desirable addition to the available tools for control of cystic echinococcosis transmission.

  11. Optimal sample storage and extraction procotols for reliable multilocus genotyping of the human parasite Schistosoma mansoni.

    PubMed

    Van den Broeck, F; Geldof, S; Polman, K; Volckaert, F A M; Huyse, T

    2011-08-01

    Genotyping individual larval stages and eggs of natural parasite populations is complicated by the difficulty of obtaining reliable genotypes from low quantity DNA template. A suitable storage and extraction protocol, together with a thorough quantification of genotyping errors are therefore crucial for molecular epidemiological studies. Here we test the robustness, handling time, ease of use, cost effectiveness and success rate of various fixation (Whatman FTA(®) Classic and Elute Cards, 70% EtOH and RNAlater(®)) and subsequent DNA extraction methods (commercial kits and proteinase K protocol). None of these methods require a cooling chain and are therefore suitable for field collection. Based on a multiplex microsatellite PCR with nine loci the success and reliability of each technique is evaluated by the proportion of samples with at least eight scored loci and the proportion of genotyping errors. If only the former is taken into account, FTA(®) Elute is recommended (83% success; 44% genotyping error; 0.2 €/sample; 1h 20 m handling time). However, when also considering the genotyping errors, handling time and ease of use, we opt for 70% EtOH with the 96-well plate technology followed by a simple proteinase K extraction (73% success; 0% genotyping error; 0.2 €/sample; 15m handling time). For eggs we suggest (1) to pool all eggs per person in 1.5 ml tubes filled with 70% EtOH for transport and (2) to identify each egg to species level prior to genotyping. To this end we extended the Rapid diagnostic PCR developed by Webster et al. (2010) with a S. mansoni-specific primer to discriminate between S. mansoni, S. haematobium and S. bovis in a single PCR reaction. The success rate of genotyping eggs was 75% (0% genotyping error). This is the first study to incorporate genotyping errors through re-amplification for the evaluation of schistosome sampling protocols and the identification of error-prone loci.

  12. Six Novel O Genotypes from Shiga Toxin-Producing Escherichia coli

    PubMed Central

    Iguchi, Atsushi; Iyoda, Sunao; Seto, Kazuko; Nishii, Hironobu; Ohnishi, Makoto; Mekata, Hirohisa; Ogura, Yoshitoshi; Hayashi, Tetsuya

    2016-01-01

    Serotyping is one of the typing techniques used to classify strains within the same species. O-serogroup diversification shows a strong association with the genetic diversity of O-antigen biosynthesis genes. In a previous study, based on the O-antigen biosynthesis gene cluster (O-AGC) sequences of 184 known Escherichia coli O serogroups (from O1 to O187), we developed a comprehensive and practical molecular O serogrouping (O genotyping) platform using a polymerase chain reaction (PCR) method, named E. coli O-genotyping PCR. Although, the validation assay using the PCR system showed that most of the tested strains were successfully classified into one of the O genotypes, it was impossible to classify 6.1% (35/575) of the strains, suggesting the presence of novel O genotypes. In this study, we conducted sequence analysis of O-AGCs from O-genotype untypeable Shiga toxin-producing E. coli (STEC) strains and identified six novel O genotypes; OgN1, OgN8, OgN9, OgN10, OgN12 and OgN31, with unique wzx and/or wzy O-antigen processing gene sequences. Additionally, to identify these novel O-genotypes, we designed specific PCR primers. A screen of O genotypes using O-genotype untypeable strains showed 13 STEC strains were classified into five novel O genotypes. The O genotyping at the molecular level of the O-AGC would aid in the characterization of E. coli isolates and will assist future studies in STEC epidemiology and phylogeny. PMID:27242776

  13. Characterization of the eg95 gene family in the G6 genotype of Echinococcus granulosus.

    PubMed

    Alvarez Rojas, Cristian A; Gauci, Charles G; Nolan, Matthew J; Harandi, Majid Fasihi; Lightowlers, Marshall W

    2012-06-01

    Cystic echinococcosis in humans and livestock animals is caused by infection with the cestode parasite Echinococcus granulosus. A number of genotypes of the parasite (designated G1-G10) are known to exist, with the genotype cluster G1-G3 and genotype G6 being responsible for the majority of humans infections. A recombinant vaccine has been developed for use in livestock to prevent infection with E. granulosus. The vaccine is based on the antigen EG95 which is expressed in the early larval stage (oncosphere) of the parasite. The EG95 antigen was originally cloned from the G1 genotype of E. granulosus and the protein has been found to be encoded by members of a small family of related genes in this genotype. Reliable information has not been available about the likely efficacy of the EG95 vaccine against genotypes other than G1. In this study, genomic DNA cloning techniques were used to characterize seven eg95-related gene fragments from the G6 genotype of E. granulosus. Three proteins appear to be encoded by these genes. Considerable differences were found between the EG95 related proteins from the G6 genotype compared with the EG95 protein from the G1 genotype. These differences suggest that the EG95-related proteins from the G6 genotype may have different antigenic epitopes compared with the current vaccine antigen. Data presented in this study have implications for future vaccine design and provide the information that would enable a G6 genotype-specific vaccine to be developed against E. granulosus, should this be considered a desirable addition to the available tools for control of cystic echinococcosis transmission. PMID:22349630

  14. UGT genotyping in belinostat dosing.

    PubMed

    Goey, Andrew K L; Figg, William D

    2016-03-01

    Certain genetic polymorphisms of UDP glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1) can reduce gene expression (*28, *60, *93) or activity (*6), thereby altering the pharmacokinetics, pharmacodynamics, and the risk of toxicities of UGT1A1 substrates, of which irinotecan is a widely-described example. This review presents an overview of the clinical effects of UGT1A1 polymorphisms on the pharmacology of UGT1A1 substrates, with a special focus on the novel histone deacetylase inhibitor belinostat. Belinostat, approved for the treatment of peripheral T-cell lymphoma, is primarily glucuronidated by UGT1A1. Recent preclinical and clinical data showed that UGT1A1*28 was associated with reduced glucuronidation in human liver microsomes, while in a retrospective analysis of a Phase I trial with patients receiving belinostat UGT1A1*60 was predominantly associated with increased belinostat plasma concentrations. Furthermore, both UGT1A1*28 and *60 variants were associated with increased incidence of thrombocytopenia and neutropenia. Using population pharmacokinetic analysis a 33% dose reduction has been proposed for patients carrying UGT1A1 variant alleles. Clinical effects of this genotype-based dosing recommendation is currently prospectively being investigated. Overall, the data suggest that UGT1A1 genotyping is useful for improving belinostat therapy. PMID:26773202

  15. Rotavirus genotypes in Belarus, 2008-2012.

    PubMed

    Semeiko, Galina V; Yermalovich, Marina A; Poliakova, Nadezhda; Mijatovic-Rustempasic, Slavica; Kerin, Tara K; Wasley, Annemarie; Videbaek, Dovile; Gentsch, Jon R; Bowen, Michael D; Samoilovich, Elena O

    2014-12-01

    This study describes group A rotavirus (RVA) genotype prevalence in Belarus from 2008 to 2012. In 2008, data from 3 sites in Belarus (Brest, Mogilev, Minsk) indicated that G4P[8] was the predominant genotype. Data from Minsk (2008-2012) showed that G4P[8] was the predominant RVA genotype in all years except in 2011 when G3P[8] was most frequently detected. Other RVA genotypes common in Europe (G1P[8], G2P[4]) were detected each year of the study. This study reveals the dominance of genotype G4P[8] in Belarus and helps to establish the baseline genotype prevalence prior to RVA vaccine introduction in the country.

  16. Absence of an essential regulatory influence of the adenovirus E1B 19-kilodalton protein on viral growth and early gene expression in human diploid WI38, HeLa, and A549 cells.

    PubMed Central

    Telling, G C; Perera, S; Szatkowski-Ozers, M; Williams, J

    1994-01-01

    Mutations in the gene encoding the adenovirus (Ad) early region 1B 19-kDa protein (the 19K gene) result in multiple phenotypic effects upon infection of permissive human cells. It has been reported, for example, that Ad type 2 (Ad2) and Ad5 with mutations in the 19K gene (19K-defective mutants) have a marked growth advantage compared with wild-type virus in human diploid WI38 cells (E. White, B. Faha, and B. Stillman, Mol. Cell. Biol. 6:3763-3773, 1986), and it was proposed that this host range phenotype stems from the large increase in viral early gene expression reported to occur in the mutant-infected cells. These observations gave rise to the hypothesis that the 19-kDa protein (the 19K protein) normally functions as a negative regulator of Ad early gene expression and growth. We have tested this hypothesis and find that Ad5 and Ad12 wild-type viruses grow as efficiently as their respective 19K-defective mutants, in1 and dl337 and pm700 and in700, in WI38 and other human cell types. Neither the accumulation of E1A cytoplasmic mRNAs nor the synthesis of E1A and other viral early proteins in these cells is altered as a result of these mutations in the 19K gene, and we conclude that the 19K protein does not play an essential role in regulating viral early gene expression or viral growth in human cells. Images PMID:8254769

  17. Targeted stock identification using multilocus genotype 'familyprinting'

    USGS Publications Warehouse

    Letcher, B.H.; King, T.L.

    1999-01-01

    We present an approach to stock identification of small, targeted populations that uses multilocus microsatellite genotypes of individual mating adults to uniquely identify first- and second-generation offspring in a mixture. We call the approach 'familyprinting'; unlike DNA fingerprinting where tissue samples of individuals are matched, offspring from various families are assigned to pairs of parents or sets of four grandparents with known genotypes. The basic unit of identification is the family, but families can be nested within a variety of stock units ranging from naturally reproducing groups of fish in a small tributary or pond from which mating adults can be sampled to large or small collections of families produced in hatcheries and stocked in specific locations. We show that, with as few as seven alleles per locus using four loci without error, first-generation offspring can be uniquely assigned to the correct family. For second-generation applications in a hatchery more alleles per locus (10) and loci (10) are required for correct assignment of all offspring to the correct set of grandparents. Using microsatellite DNA variation from an Atlantic salmon (Salmo solar) restoration river (Connecticut River, USA), we also show that this population contains sufficient genetic diversity in sea-run returns for 100% correct first, generation assignment and 97% correct second-generation assignment using 14 loci. We are currently using first- and second-generation familyprinting in this population with the ultimate goal of identifying stocking tributary. In addition to within-river familyprinting, there also appears to be sufficient genetic diversity within and between Atlantic salmon populations for identification of 'familyprinted' fish in a mixture of multiple populations. We also suggest that second-generation familyprinting with multiple populations may also provide a tool for examining stock structure. Familyprinting with microsatellite DNA markers is a viable

  18. Predicted redistribution of Ceratomyxa shasta genotypes with salmonid passage in the Deschutes River, Oregon.

    PubMed

    Stinson, Matthew E T; Bartholomew, Jerri L

    2012-12-01

    A series of dams on the Deschutes River, Oregon, act as migration barriers that segregate the river system into upper and lower basins. Proposed fish passage between basins would reunite populations of native potamodromous fish and allow anadromous fish of Deschutes River origin access to the upper basin. We assessed the potential redistribution of host-species-specific genotypes (O, I, II, III) of the myxozoan parasite Ceratomyxa shasta that could occur with fish passage and examined the influence of nonnative fish on genotype composition. To determine the present distribution of the parasite genotypes, we exposed eight salmonid species-three native and five stocked for sport fishing-in present and predicted anadromous salmonid habitats. We monitored fish for infection by C. shasta and sequenced a section of the parasite ribosomal DNA gene from fish and water samples to determine parasite genotype. Genotype O was present in both upper and lower basins and detected only in steelhead Oncorhynchus mykiss. Genotype I was spatially limited to the lower basin, isolated predominantly from Chinook salmon O. tshawytscha, and lethal for this species only. Genotype II was detected in both basins and in multiple species, but only as a minor component of the infection. Genotype III was also present in both basins, had a wide host range, and caused mortality in native steelhead and multiple nonnative species. Atlantic salmon Salmo salar and kokanee O. nerka were the least susceptible to infection by any genotype of C. shasta. Our findings confirmed the host-specific patterns of C. shasta infections and indicated that passage of Chinook salmon would probably spread genotype I into the upper Deschutes River basin, but with little risk to native salmonid populations. PMID:23146111

  19. Prevalence and genotype distribution of rotaviruses in children with gastroenteritis in Rize province.

    PubMed

    Dereci, Selim; Çopur Çiçek, Ayşegül; Savaş Acar, Sümeyra; Bakkaloğlu, Zekiye; Özkasap, Serdar; Kanber, Kadri; Hacisalihoğlu, Şadan; Albayrak, Yücehan; Durmaz, Rıza

    2015-01-01

    Determination of the distribution of rotavirus genotypes is essential for understanding the epidemiology of this virus responsible for nearly half a million of deaths in patients with gastroenteritis worldwide. In the present study, we aimed to genotype the rotavirus strains isolated from diarrheal stool samples in children under 5 years old. A total of 1297 fecal samples were collected, and rotavirus antigen was detected in 73 of these samples. Antigen-positive samples were transferred to the Public Health Agency of Turkey, Molecular Microbiology Research Laboratory, and were tested for determination of genotypes G and P using semi-nested multiplex polymerase chain reaction method performed with consensus- and genotype-specific primers. Twelve specimens were found to be negative for rotavirus in genotyping method. All the positive-strains were in G1-4, G8-9, P(4), P(8), and P(9) genotypes. The most frequent GP genotype combinations were found to be G9P(8) in 21 strains (34.4%), G2P(4) in 14 strains (23.0%), and G1P(8) in 12 strains (19.7%). We found 10 distinct genotypes amongst a total of 61 strains. Among the strains isolated and genotyped in our study, 90.2% (55/61) and 67.2% (41/61) have already been included in the two existing commercial vaccines. In conclusion, these findings implicate the necessity of development of region-specific vaccines after evaluation of the local genotype distribution. Further studies on the large number of rotavirus strains would contribute to this process. PMID:26295292

  20. [Study on RHCE genotyping of Rh blood group in Uygur nationality of Xinjiang in China].

    PubMed

    Bai, Xu-Hua; Lan, Jiong-Cai; Gong, Xiao-Yan; Cui, Li; Zhou, Hua-You

    2010-10-01

    This study was aimed to investigate the characteristics of RHCE genotyping of Xinjiang Uygur nationality population in China. Primers for detecting RHCE genes were designed according to the references, 89 Uygur nationality RhD-negative samples, 233 Han nationality RhD-negative samples and 109 Han nationality RhD-positive samples were detected by sequence-specific primer-polymerase chain reaction (SSP-PCR) for RHCE genotyping. All above-mentioned samples were unrelated. The results indicated that RHE/e genotyping results were consistent with the serological test results in the samples of Uygur and Han nationality, regardless of the RhD-negative samples or the RhD-positive samples. The RHC/c genotyping results from 89 RhD-negative samples of Uygur nationality were consistent with serological test results. However, total error of RHC/c genotyping from 233 RhD-negative and 109 RhD-positive samples of Han nationality was 5.05%. In conclusion, this method of RHCE genotyping is suitable for the analysis of the RHE/e genotyping of Uygur nationality, no erroneous RHC/c genotyping of Uygur nationality was found in this study, but this method needs to be further studied.

  1. Effect of within-species plant genotype mixing on habitat preference of a polyphagous insect predator.

    PubMed

    Ninkovic, Velemir; Al Abassi, Sate; Ahmed, Elham; Glinwood, Robert; Pettersson, Jan

    2011-06-01

    The effects of within-species plant genotype mixing on the habitat preference of a polyphagous ladybird were studied. Plant species diversity is often claimed to positively affect habitat preferences of insect predators, but the effects of within-species genotype diversity have not been extensively studied. In a field experiment with different barley (Hordeum vulgare) genotypes in mixed and pure stands, adult seven-spot ladybird Coccinella septempunctata, a polyphagous predator, preferred a specific combination of genotypes over the single genotypes alone before aphids had arrived in the crop, and again when aphids were emigrating. In laboratory experiments on adult ladybird orientation to odour from barley, ladybirds were attracted/arrested by the mixed odour of the same barley genotype mixture that was preferred in the field. Exposure of one barley genotype to volatiles from the other also caused the odour of the exposed plants to become more attractive to ladybirds. The results support the hypothesis that plant volatiles may attract or arrest foraging adult ladybirds, contributing to the selection of favourable habitats, and they show that within-species plant genotype mixing can shape interactions within multitrophic communities.

  2. Pneumocystis jirovecii multilocus genotyping profiles in patients from Portugal and Spain.

    PubMed

    Esteves, F; Montes-Cano, M A; de la Horra, C; Costa, M C; Calderón, E J; Antunes, F; Matos, O

    2008-04-01

    Pneumonia caused by the opportunistic organism Pneumocystis jirovecii is a clinically important infection affecting AIDS and other immunocompromised patients. The present study aimed to compare and characterise the frequency pattern of DNA sequences from the P. jirovecii mitochondrial large-subunit rRNA (mtLSU rRNA) gene, the dihydropteroate synthase (DHPS) gene and the internal transcribed spacer (ITS) regions of the nuclear rRNA operon in specimens from Lisbon (Portugal) and Seville (Spain). Total DNA was extracted and used for specific molecular sequence analysis of the three loci. In both populations, mtLSU rRNA gene analysis revealed an overall prevalence of genotype 1. In the Portuguese population, genotype 2 was the second most common, followed by genotype 3. Inversely, in the Spanish population, genotype 3 was the second most common, followed by genotype 2. The DHPS wild-type sequence was the genotype observed most frequently in both populations, and the DHPS genotype frequency pattern was identical to distribution patterns revealed in other European studies. ITS types showed a significant diversity in both populations because of the high sequence variability in these genomic regions. The most prevalent ITS type in the Portuguese population was Eg, followed by Cg. In contrast to other European studies, Bi was the most common ITS type in the Spanish samples, followed by Eg. A statistically significant association between mtLSU rRNA genotype 1 and ITS type Eg was revealed.

  3. Potential antioxidant response to coffee — A matter of genotype?

    PubMed Central

    Hassmann, Ute; Haupt, Larisa M.; Smith, Robert A.; Winkler, Swantje; Bytof, Gerhard; Lantz, Ingo; Griffiths, Lyn R.; Marko, Doris

    2014-01-01

    In a human intervention study, coffee combining natural green coffee bean constituents and dark roast products was identified as a genotype-dependent inducer of the Nrf2/ARE pathway, significantly affecting Nrf2 gene expression and downstream GST1A1 and UGT1A1 gene transcription. The observed transcriptional changes correlated with the presence of specific Nrf2 genotypes suggesting their influence on both Nrf2 and subsequent ARE-dependent GST1A1 and UGT1A1 transcription. While the presence of the − 653 SNP seems to be advantageous, resulting in higher Nrf2, GST1A1 and UGT1A1 gene transcription following coffee consumption, in contrast, the presence of the − 651 SNP significantly down-regulated the response to the study coffee. Furthermore, the presence of the B/B genotype in GST1A1 along with the frequency of the [TA]6/6 and [TA]7/7 polymorphisms in UGT1A1 appeared to significantly increase sensitivity toward coffee-induced gene transcription. This data suggests that when examining the role of the Nrf2/ARE pathway in the regulation of antioxidative and chemopreventive phase II efficacy, individual genotypes should be included when considering the potency of bioactive food/food constituents and their therapeutic potential. PMID:25606436

  4. Potential antioxidant response to coffee - A matter of genotype?

    PubMed

    Hassmann, Ute; Haupt, Larisa M; Smith, Robert A; Winkler, Swantje; Bytof, Gerhard; Lantz, Ingo; Griffiths, Lyn R; Marko, Doris

    2014-12-01

    In a human intervention study, coffee combining natural green coffee bean constituents and dark roast products was identified as a genotype-dependent inducer of the Nrf2/ARE pathway, significantly affecting Nrf2 gene expression and downstream GST1A1 and UGT1A1 gene transcription. The observed transcriptional changes correlated with the presence of specific Nrf2 genotypes suggesting their influence on both Nrf2 and subsequent ARE-dependent GST1A1 and UGT1A1 transcription. While the presence of the - 653 SNP seems to be advantageous, resulting in higher Nrf2, GST1A1 and UGT1A1 gene transcription following coffee consumption, in contrast, the presence of the - 651 SNP significantly down-regulated the response to the study coffee. Furthermore, the presence of the B/B genotype in GST1A1 along with the frequency of the [TA]6/6 and [TA]7/7 polymorphisms in UGT1A1 appeared to significantly increase sensitivity toward coffee-induced gene transcription. This data suggests that when examining the role of the Nrf2/ARE pathway in the regulation of antioxidative and chemopreventive phase II efficacy, individual genotypes should be included when considering the potency of bioactive food/food constituents and their therapeutic potential. PMID:25606436

  5. Molecular genotyping and quantitation assay for rotavirus surveillance.

    PubMed

    Liu, Jie; Lurain, Kate; Sobuz, Shihab U; Begum, Sharmin; Kumburu, Happiness; Gratz, Jean; Kibiki, Gibson; Toney, Denise; Gautam, Rashi; Bowen, Michael D; Petri, William A; Haque, Rashidul; Houpt, Eric R

    2015-03-01

    Rotavirus genotyping is useful for surveillance purposes especially in areas where rotavirus vaccination has been or will be implemented. RT-PCR based molecular methods have been applied widely, but quantitative assays targeting a broad spectrum of genotypes have not been developed. Three real time RT-PCR panels were designed to identify G1, G2, G9, G12 (panel GI), G3, G4, G8, G10 (panel GII), and P[4], P[6], P[8], P[10], P[11] (panel P), respectively. An assay targeting NSP3 was included in both G panels as an internal control. The cognate assays were also formulated as one RT-PCR-Luminex panel for simultaneous detection of all the genotypes listed above plus P[9]. The assays were evaluated with various rotavirus isolates and 89 clinical samples from Virginia, Bangladesh and Tanzania, and exhibited 95% (81/85) sensitivity compared with the conventional RT-PCR-Gel-electrophoresis method, and 100% concordance with sequencing. Real time assays identified a significantly higher rate of mixed genotypes in Bangladeshi samples than the conventional gel-electrophoresis-based RT-PCR assay (32.5% versus 12.5%, P<0.05). In these mixed infections, the relative abundance of the rotavirus types could be estimated by Cq values. These typing assays detect and discriminate a broad range of G/P types circulating in different geographic regions with high sensitivity and specificity and can be used for rotavirus surveillance.

  6. Potential antioxidant response to coffee - A matter of genotype?

    PubMed

    Hassmann, Ute; Haupt, Larisa M; Smith, Robert A; Winkler, Swantje; Bytof, Gerhard; Lantz, Ingo; Griffiths, Lyn R; Marko, Doris

    2014-12-01

    In a human intervention study, coffee combining natural green coffee bean constituents and dark roast products was identified as a genotype-dependent inducer of the Nrf2/ARE pathway, significantly affecting Nrf2 gene expression and downstream GST1A1 and UGT1A1 gene transcription. The observed transcriptional changes correlated with the presence of specific Nrf2 genotypes suggesting their influence on both Nrf2 and subsequent ARE-dependent GST1A1 and UGT1A1 transcription. While the presence of the - 653 SNP seems to be advantageous, resulting in higher Nrf2, GST1A1 and UGT1A1 gene transcription following coffee consumption, in contrast, the presence of the - 651 SNP significantly down-regulated the response to the study coffee. Furthermore, the presence of the B/B genotype in GST1A1 along with the frequency of the [TA]6/6 and [TA]7/7 polymorphisms in UGT1A1 appeared to significantly increase sensitivity toward coffee-induced gene transcription. This data suggests that when examining the role of the Nrf2/ARE pathway in the regulation of antioxidative and chemopreventive phase II efficacy, individual genotypes should be included when considering the potency of bioactive food/food constituents and their therapeutic potential.

  7. Additional novel Cryptosporidium genotypes in ornamental fishes.

    PubMed

    Morine, M; Yang, R; Ng, J; Kueh, S; Lymbery, A J; Ryan, U M

    2012-12-21

    Current knowledge on the prevalence and genotypes of Cryptosporidium in fishes is still limited. This study investigated the prevalence of Cryptosporidium species in 171 ornamental fishes, belonging to 33 species, collected from 8 commercial aquariums around Perth, Western Australia. All samples were screened by nested PCR targeting the 18S rRNA locus. A total of 6 positives were identified by PCR at the 18S locus from 4 different species of fishes (red eye tetra, Moenkhausia sanctaefilomenae; gold gourami, Trichogaster trichopterus; neon tetra, Paracheirodon innesi; goldfish, Carassius auratus auratus), giving an overall prevalence of 3.5% (6/171). Four different genotypes were identified, only one of which has been previously reported in fish; piscine genotype 4 in a neon tetra isolate, a rat genotype III-like isolate in a goldfish, a novel genotype in three isolates from red eye (piscine genotype 7) which exhibited a 3.5% genetic distance from piscine genotype 1 and a piscine genotype 6-like from a gold gourami (1% genetic distance). Further biological and genetic characterisation is required to determine the relationship of these genotypes to established species and strains of Cryptosporidium. PMID:22819587

  8. Using DNA pools for genotyping trios.

    PubMed

    Beckman, Kenneth B; Abel, Kenneth J; Braun, Andreas; Halperin, Eran

    2006-01-01

    The genotyping of mother-father-child trios is a very useful tool in disease association studies, as trios eliminate population stratification effects and increase the accuracy of haplotype inference. Unfortunately, the use of trios for association studies may reduce power, since it requires the genotyping of three individuals where only four independent haplotypes are involved. We describe here a method for genotyping a trio using two DNA pools, thus reducing the cost of genotyping trios to that of genotyping two individuals. Furthermore, we present extensions to the method that exploit the linkage disequilibrium structure to compensate for missing data and genotyping errors. We evaluated our method on trios from CEPH pedigree 66 of the Coriell Institute. We demonstrate that the error rates in the genotype calls of the proposed protocol are comparable to those of standard genotyping techniques, although the cost is reduced considerably. The approach described is generic and it can be applied to any genotyping platform that achieves a reasonable precision of allele frequency estimates from pools of two individuals. Using this approach, future trio-based association studies may be able to increase the sample size by 50% for the same cost and thereby increase the power to detect associations.

  9. Genotyping of Mycobacterium tuberculosis: application in epidemiologic studies

    PubMed Central

    Kato-Maeda, Midori; Metcalfe, John Z.; Flores, Laura

    2014-01-01

    Genotyping is used to track specific isolates of Mycobacterium tuberculosis in a community. It has been successfully used in epidemiologic research (termed ‘molecular epidemiology’) to study the transmission dynamics of TB. In this article, we review the genetic markers used in molecular epidemiologic studies including the use of whole-genome sequencing technology. We also review the public health application of molecular epidemiologic tools. PMID:21366420

  10. Detection and differentiation of Japanese encephalitis virus genotype I and genotype III by reverse transcription loop-mediated isothermal amplification combined with restriction fragment length polymorphism.

    PubMed

    Zhang, Liang; Cao, Sanjie; Wu, Rui; Zhu, Shuquan; Liu, Hanyang; Yuan, Lei; Shi, Shuangyan; Zhang, Dan; Huang, Xiaobo; Wen, Xintian; Wen, Yiping; Yan, Qigui; Huang, Yong; Ma, Xiaoping

    2015-04-01

    Japanese encephalitis (JE), which is a mosquito-borne arboviral infection, is the leading cause of viral encephalitis in Asian countries. The causative agent of JE is Japanese encephalitis virus (JEV), in which the predominant genotype has changed from genotype III (G III) to genotype I (G I). However, a method for the rapid differentiation between JEV G I and G III remains unavailable. This study aimed to establish a rapid JEV genotyping method using reverse transcription loop-mediated isothermal amplification (RT-LAMP). An Spe I site, which was located in the target sequence (C gene) of JEV G III strains but not in JEV G I strains, was selected as the RT-LAMP target. After testing 64 specimens, results showed that RT-LAMP can detect and differentiate JEV G I and G III specifically. Thus, a novel RT-LAMP system for the rapid detection and differentiation of JEV G I and G III was developed successfully.

  11. Validation of a blood group genotyping method based on high-resolution melting curve analysis.

    PubMed

    Gong, Tianxiang; Hong, Ying; Wang, Naihong; Fu, Xuemei; Zhou, Changhua

    2014-01-01

    The detection of polymorphism is the basis of blood group genotyping and phenotype prediction. Genotyping may be useful to determine blood groups when serologic results are unclear. The development and application of different methods for blood group genotyping may be needed as a substitute for blood group typing. The purpose of this study is to establish an approach for blood group genotyping based on a melting curve analysis of real-time polymerase chain reaction (PCR). Using DNA extracted from whole blood, we developed and validated a DNA typing method for detecting DO*01/DO*02, DO*01/DI*02, LU*01/LU*02, and GYPB*03/GYBP*04 alleles using a melting curve analysis. All assays were confirmed with a commercial reagent containing sequence-specific primers (PCR-SSP), and a cohort of the samples was confirmed with sequencing. Results for all blood groups were within the range of specificity and assay variability. Genotypes of 300 blood donors were fully consistent with PCR-SSP data. The obtained genotype distribution is in complete concordance with existing data for the Chinese population. There are several advantages for this approach of blood group genotyping: lower contamination rates with PCR products in this laboratory, ease of performance, automation potential, and rapid cycling time.

  12. SNP genotyping by heteroduplex analysis.

    PubMed

    Paniego, Norma; Fusari, Corina; Lia, Verónica; Puebla, Andrea

    2015-01-01

    Heteroduplex-based genotyping methods have proven to be technologically effective and economically efficient for low- to medium-range throughput single-nucleotide polymorphism (SNP) determination. In this chapter we describe two protocols that were successfully applied for SNP detection and haplotype analysis of candidate genes in association studies. The protocols involve (1) enzymatic mismatch cleavage with endonuclease CEL1 from celery, associated with fragment separation using capillary electrophoresis (CEL1 cleavage), and (2) differential retention of the homo/heteroduplex DNA molecules under partial denaturing conditions on ion pair reversed-phase liquid chromatography (dHPLC). Both methods are complementary since dHPLC is more versatile than CEL1 cleavage for identifying multiple SNP per target region, and the latter is easily optimized for sequences with fewer SNPs or small insertion/deletion polymorphisms. Besides, CEL1 cleavage is a powerful method to localize the position of the mutation when fragment resolution is done using capillary electrophoresis.

  13. minimac2: faster genotype imputation

    PubMed Central

    Fuchsberger, Christian; Abecasis, Gonçalo R.; Hinds, David A.

    2015-01-01

    Summary: Genotype imputation is a key step in the analysis of genome-wide association studies. Upcoming very large reference panels, such as those from The 1000 Genomes Project and the Haplotype Consortium, will improve imputation quality of rare and less common variants, but will also increase the computational burden. Here, we demonstrate how the application of software engineering techniques can help to keep imputation broadly accessible. Overall, these improvements speed up imputation by an order of magnitude compared with our previous implementation. Availability and implementation: minimac2, including source code, documentation, and examples is available at http://genome.sph.umich.edu/wiki/Minimac2 Contact: cfuchsb@umich.edu, goncalo@umich.edu PMID:25338720

  14. Genotyping of strawberry (Fragaria x ananassa Duch.) cultivars by DNA markers: interlaboratory study.

    PubMed

    Kunihisa, Miyuki; Ueda, Hiroshi; Fukino, Nobuko; Matsumoto, Satoru

    2009-01-01

    Fourteen Japanese laboratories validated the reproducibility of genotyping by 25 cleavage amplified polymorphic sequence (CAPS) markers for discrimination of strawberry (Fragaria x ananassa Duch.) cultivars. Both the sensitivity and specificity rate of 12 markers were 100%, those of another 12 were >95%, and those of 1 were >90%. These results indicate that the method of genotyping by the CAPS markers was highly reproducible and could provide a useful basis for practical identification of strawberry cultivars. This is the first report of the statistical validation of crop genotyping by DNA markers.

  15. Chikungunya Outbreaks Caused by African Genotype, India

    PubMed Central

    Yergolkar, Prasanna N.; Tandale, Babasaheb V.; Arankalle, Vidya A.; Sathe, Padmakar S.; Gandhe, Swati S.; Gokhle, Mangesh D.; Jacob, George P.; Hundekar, Supriya L.

    2006-01-01

    Chikungunya fever is reported in India after 32 years. Immunoglobulin M antibodies and virus isolation confirmed the cause. Phylogenic analysis based on partial sequences of NS4 and E1 genes showed that all earlier isolates (1963–1973) were Asian genotype, whereas the current and Yawat (2000) isolates were African genotype. PMID:17176577

  16. Disentangling pooled triad genotypes for association studies.

    PubMed

    Shi, Min; Umbach, David M; Weinberg, Clarice R

    2014-09-01

    Association studies that genotype affected offspring and their parents (triads) offer robustness to genetic population structure while enabling assessments of maternal effects, parent-of-origin effects, and gene-by-environment interaction. We propose case-parents designs that use pooled DNA specimens to make economical use of limited available specimens. One can markedly reduce the number of genotyping assays required by randomly partitioning the case-parent triads into pooling sets of h triads each and creating three pools from every pooling set, one pool each for mothers, fathers, and offspring. Maximum-likelihood estimation of relative risk parameters proceeds via log-linear modeling using the expectation-maximization algorithm. The approach can assess offspring and maternal genetic effects and accommodate genotyping errors and missing genotypes. We compare the power of our proposed analysis for testing offspring and maternal genetic effects to that based on a difference approach and that of the gold standard based on individual genotypes, under a range of allele frequencies, missing parent proportions, and genotyping error rates. Power calculations show that the pooling strategies cause only modest reductions in power if genotyping errors are low, while reducing genotyping costs and conserving limited specimens.

  17. Using genotypic information to reduce disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this presentation is to provide a cursory overview of how genotypic data may be utilized by veterinarians in the future. Genotypic information is accumulating at a rapid pace. This information may reveal deleterious genes, quantitative trait loci, and genetic predisposition for a ...

  18. Filling in missing genotypes using haplotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Unknown genotypes can be made known (imputed) from observed genotypes at the same or nearby loci of relatives using pedigree haplotyping, or from matching allele patterns (regardless of pedigree) using population haplotyping. Fortran program findhap.f90 was designed to combine population and pedigre...

  19. New cryptosporidium genotypes in HIV-infected persons.

    PubMed Central

    Pieniazek, N. J.; Bornay-Llinares, F. J.; Slemenda, S. B.; da Silva, A. J.; Moura, I. N.; Arrowood, M. J.; Ditrich, O.; Addiss, D. G.

    1999-01-01

    Using DNA sequencing and phylogenetic analysis, we identified four distinct Cryptosporidium genotypes in HIV-infected patients: genotype 1 (human), genotype 2 (bovine) Cryptosporidium parvum, a genotype identical to C. felis, and one identical to a Cryptosporidium sp. isolate from a dog. This is the first identification of human infection with the latter two genotypes. PMID:10341184

  20. Virulence correlates with fitness in vivo for two M group genotypes of Infectious hematopoietic necrosis virus (IHNV).

    USGS Publications Warehouse

    Wargo, Andrew R.; Garver, Kyle A.; Kurath, Gael

    2010-01-01

    The nature of the association between viral fitness and virulence remains elusive in vertebrate virus systems, partly due to a lack of in vivo experiments using statistically sufficient numbers of replicate hosts. We examined the relationship between virulence and fitness in Infectious hematopoietic necrosis virus (IHNV), in vivo, in intact living rainbow trout. Trout were infected with a high or low virulence genotype of M genogroup IHNV, or a mixture of the two genotypes, so as to calculate relative fitness and the effect of a competition environment on fitness. Fitness was measured as total viral load in the host at time of peak viral density, quantified by genotype-specific quantitative RT-PCR (qRT-PCR). The more virulent IHNV genotype reached higher densities in both single and mixed infections. There was no effect of competition on the performance of either genotype. Our results suggest a positive link between IHNV genotype fitness and virulence.

  1. LinkImpute: Fast and Accurate Genotype Imputation for Nonmodel Organisms.

    PubMed

    Money, Daniel; Gardner, Kyle; Migicovsky, Zoë; Schwaninger, Heidi; Zhong, Gan-Yuan; Myles, Sean

    2015-11-01

    Obtaining genome-wide genotype data from a set of individuals is the first step in many genomic studies, including genome-wide association and genomic selection. All genotyping methods suffer from some level of missing data, and genotype imputation can be used to fill in the missing data and improve the power of downstream analyses. Model organisms like human and cattle benefit from high-quality reference genomes and panels of reference genotypes that aid in imputation accuracy. In nonmodel organisms, however, genetic and physical maps often are either of poor quality or are completely absent, and there are no panels of reference genotypes available. There is therefore a need for imputation methods designed specifically for nonmodel organisms in which genomic resources are poorly developed and marker order is unreliable or unknown. Here we introduce LinkImpute, a software package based on a k-nearest neighbor genotype imputation method, LD-kNNi, which is designed for unordered markers. No physical or genetic maps are required, and it is designed to work on unphased genotype data from heterozygous species. It exploits the fact that markers useful for imputation often are not physically close to the missing genotype but rather distributed throughout the genome. Using genotyping-by-sequencing data from diverse and heterozygous accessions of apples, grapes, and maize, we compare LD-kNNi with several genotype imputation methods and show that LD-kNNi is fast, comparable in accuracy to the best-existing methods, and exhibits the least bias in allele frequency estimates.

  2. LinkImpute: Fast and Accurate Genotype Imputation for Nonmodel Organisms

    PubMed Central

    Money, Daniel; Gardner, Kyle; Migicovsky, Zoë; Schwaninger, Heidi; Zhong, Gan-Yuan; Myles, Sean

    2015-01-01

    Obtaining genome-wide genotype data from a set of individuals is the first step in many genomic studies, including genome-wide association and genomic selection. All genotyping methods suffer from some level of missing data, and genotype imputation can be used to fill in the missing data and improve the power of downstream analyses. Model organisms like human and cattle benefit from high-quality reference genomes and panels of reference genotypes that aid in imputation accuracy. In nonmodel organisms, however, genetic and physical maps often are either of poor quality or are completely absent, and there are no panels of reference genotypes available. There is therefore a need for imputation methods designed specifically for nonmodel organisms in which genomic resources are poorly developed and marker order is unreliable or unknown. Here we introduce LinkImpute, a software package based on a k-nearest neighbor genotype imputation method, LD-kNNi, which is designed for unordered markers. No physical or genetic maps are required, and it is designed to work on unphased genotype data from heterozygous species. It exploits the fact that markers useful for imputation often are not physically close to the missing genotype but rather distributed throughout the genome. Using genotyping-by-sequencing data from diverse and heterozygous accessions of apples, grapes, and maize, we compare LD-kNNi with several genotype imputation methods and show that LD-kNNi is fast, comparable in accuracy to the best-existing methods, and exhibits the least bias in allele frequency estimates. PMID:26377960

  3. Chronic hepatitis C virus infections in brazilian patients: association with genotypes, clinical parameters and response to long term alpha interferon therapy

    PubMed

    Bassit; Da Silva LC; Ribeiro-Dos-Santos; Maertens; Carrilho; Fonseca; Alves; Gayotto; Pereira; Takei; Chamone; Saez-Alquezar

    1999-05-01

    The present study assessed the clinical significance of hepatitis C virus (HCV) genotypes and their influence on response to long term recombinant-interferon-alpha (r-IFN-alpha) therapy in Brazilian patients. One hundred and thirty samples from patients previously genotyped for the HCV and with histologically confirmed chronic hepatitis C (CH-C) were evaluated for clinical and epidemiological parameters (sex, age, time of HCV infection and transmission routes). No difference in disease activity, sex, age or mode and time of transmission were seen among patients infected with HCV types 1, 2 or 3. One hundred and thirteen of them were treated with 3 million units of r-IFN-alpha, 3 times a week for 12 months. Initial response (IR) was significantly better in patients with genotype 2 (100%) and 3 (46%) infections than in patients with genotype 1 (29%) (p < 0. 005). Among subtypes, difference in IR was observed between 1b and 2 (p < 0.005), and between 1b and 3a (p < 0.05). Sustained response (SR) was observed in 12% for (sub)type 1a, 13% for 1b, 19% for 3a, and 40% for type 2; significant differences were found between 1b and 2 (p < 0.001), and between 1b and 3a (p < 0.05). Moreover, presence of cirrhosis was significantly associated with non response and response with relapse (p < 0.05). In conclusion, non-1 HCV genotype and lack of histological diagnosis of cirrhosis were the only baseline features associated with sustained response to treatment. These data indicate that HCV genotyping may have prognostic relevance in the responsiveness to r-IFN-alpha therapy in Brazilian patients with chronic HCV infection, as seen in other reports worldwide. PMID:10529839

  4. Toward fully automated genotyping: Genotyping microsatellite markers by deconvolution

    SciTech Connect

    Perlin, M.W.; Lancia, G.; See-Kiong, Ng

    1995-11-01

    Dense genetic linkage maps have been constructed for the human and mouse genomes, with average densities of 2.9 cM and 0.35 cM, respectively. These genetic maps are crucial for mapping both Mendelian and complex traits and are useful in clinical genetic diagnosis. Current maps are largely comprised of abundant, easily assayed, and highly polymorphic PCR-based microsatellite markers, primarily dinucleotide (CA){sub n} repeats. One key limitation of these length polymorphisms is the PCR stutter (or slippage) artifact that introduces additional stutter bands. With two (or more) closely spaced alleles, the stutter bands overlap, and it is difficult to accurately determine the correct alleles; this stutter phenomenon has all but precluded full automation, since a human must visually inspect the allele data. We describe here novel deconvolution methods for accurate genotyping that mathematically remove PCR stutter artifact from microsatellite markers. These methods overcome the manual interpretation bottleneck and thereby enable full automation of genetic map construction and use. New functionalities, including the pooling of DNAs and the pooling of markers, are described that may greatly reduce the associated experimentation requirements. 32 refs., 5 figs., 3 tabs.

  5. Differences in ovarian aging patterns between races are associated with ovarian genotypes and sub-genotypes of the FMR1 gene

    PubMed Central

    2012-01-01

    Background Ovarian aging patterns differ between races, and appear to affect fertility treatment outcomes. What causes these differences is, however, unknown. Variations in ovarian aging patterns have recently been associated with specific ovarian genotypes and sub-genotypes of the FMR1 gene. We, therefore, attempted to determine differences in how functional ovarian reserve (FOR) changes with advancing age between races, and whether changes are associated with differences in distribution of ovarian genotypes and sub-genotypes of the FMR1 gene. Methods We determined in association with in vitro fertilization (IVF) FOR in 62 young Caucasian, African and Asian oocyte donors and 536 older infertility patients of all three races, based on follicle stimulating hormone (FSH), anti-Müllerian hormone (AMH) and oocyte yields, and investigated whether differences between races are associated with differences in distribution of FMR1 genotypes and sub-genotypes. Results Changes in distribution of mean FSH, AMH and oocyte yields between young donors and older infertility patients were significant (all P < 0.001). Donors did not demonstrate significant differences between races in AMH and FSH but demonstrated significant differences in oocyte yields [F(2,59) = 4.22, P = 0.019]: Specifically, African donors demonstrated larger oocyte yields than Caucasians (P = 0.008) and Asians (P = 0.022). In patients, AMH levels differed significantly between races [F (2,533) = 4.25, P = 0.015]. Holm-Sidak post-hoc comparisons demonstrated that Caucasians demonstrated lower AMH in comparison to Asians (P = 0.007). Percentages of FMR1 genotypes and sub-genotypes in patients varied significantly between races, with Asians demonstrating fewer het-norm/low sub-genotypes than Caucasians and Africans (P = 0.012). Conclusion FOR changes in different races at different rates, and appears to parallel ovarian FMR1 genotypes and sub-genotype distributions. Differences

  6. Variation in stress resistance patterns among stx genotypes and genetic lineages of shiga toxin-producing Escherichia coli O157.

    PubMed

    Lee, Ken-Ichi; French, Nigel P; Jones, Geoff; Hara-Kudo, Yukiko; Iyoda, Sunao; Kobayashi, Hideki; Sugita-Konishi, Yoshiko; Tsubone, Hirokazu; Kumagai, Susumu

    2012-05-01

    To evaluate the relationship between bacterial genotypes and stress resistance patterns, we exposed 57 strains of Shiga toxin-producing Escherichia coli (STEC) O157 to acid, freeze-thaw, heat, osmotic, oxidative, and starvation stresses. Inactivation rates were calculated in each assay and subjected to univariate and multivariate analyses, including principal component analysis (PCA) and cluster analysis. The stx genotype was determined for each strain as was the lineage-specific polymorphism assay (LSPA6) genotype. In univariate analyses, strains of the stx(1) stx(2) genotype showed greater resistance to heat than strains of the stx(1) stx(2c) genotype; moreover, strains of the stx(1) stx(2) genotype showed greater resistance to starvation than strains of the stx(2) or stx(2c) genotypes. LSPA6 lineage I (LI) strains showed greater resistance to heat and starvation than LSPA6 lineage II (LII) strains. PCA revealed a general trend that a strain with greater resistance to one type of stress tended to have greater resistance to other types of stresses. In cluster analysis, STEC O157 strains were grouped into stress-resistant, stress-sensitive, and intermediate clusters. In stx genotypes, all strains of the stx(1) stx(2) genotype were grouped with the stress-resistant cluster, whereas 72.7% (8/11) of strains of the stx(1) stx(2c) genotype grouped with the stress-sensitive cluster. In LI strains, 77.8% (14/18) of the strains were grouped with the stress-resistant cluster, whereas 64.7% (11/17) of LII strains were grouped with the stress-sensitive cluster. These results indicate that the genotypes of STEC O157 that are frequently associated with human illness, i.e., LI or the stx(1) stx(2) genotype, have greater multiple stress resistance than do strains of other genotypes.

  7. Dysbindin-1 genotype effects on emotional working memory

    PubMed Central

    Wolf, Claudia; Jackson, Margaret C.; Kissling, Christian; Thome, Johannes; Linden, David E.J.

    2009-01-01

    We combined functional imaging and genetics to investigate the behavioral and neural effects of a dysbindin-1 (DTNBP1) genotype associated with the expression level of this important synaptic protein, which has been implicated in schizophrenia. On a working memory (WM) task for emotional faces, participants with the genotype related to increased expression showed higher WM capacity for happy faces compared to the genotype related to lower expression. Activity in several task-related brain areas with known DTNBP1 expression was increased, including hippocampus, temporal and frontal cortex. Although these increases occurred across emotions, they were mostly observed in areas whose activity correlated with performance for happy faces. This suggests effects of variability in DTNBP1 on emotion-specific WM capacity and region-specific task-related brain activation in humans. Synaptic effects of DTNBP1 implicate that altered dopaminergic and/or glutamatergic neurotransmission may be related to the increased WM capacity. The combination of imaging and genetics thus allows us to bridge the gap between the cellular/molecular and systems/behavioral level and extend the cognitive neuroscience approach to a comprehensive biology of cognition. PMID:20010894

  8. Daphnia parasite dynamics across multiple Caullerya epidemics indicate selection against common parasite genotypes.

    PubMed

    González-Tortuero, Enrique; Rusek, Jakub; Turko, Patrick; Petrusek, Adam; Maayan, Inbar; Piálek, Lubomír; Tellenbach, Christoph; Gießler, Sabine; Spaak, Piet; Wolinska, Justyna

    2016-08-01

    Studies of parasite population dynamics in natural systems are crucial for our understanding of host-parasite coevolutionary processes. Some field studies have reported that host genotype frequencies in natural populations change over time according to parasite-driven negative frequency-dependent selection. However, the temporal patterns of parasite genotypes have rarely been investigated. Moreover, parasite-driven negative frequency-dependent selection is contingent on the existence of genetic specificity between hosts and parasites. In the present study, the population dynamics and host-genotype specificity of the ichthyosporean Caullerya mesnili, a common endoparasite of Daphnia water fleas, were analysed based on the observed sequence variation in the first internal transcribed spacer (ITS1) of the ribosomal DNA. The Daphnia population of lake Greifensee (Switzerland) was sampled and subjected to parasite screening and host genotyping during C. mesnili epidemics of four consecutive years. The ITS1 of wild-caught C. mesnili-infected Daphnia was sequenced using the 454 pyrosequencing platform. The relative frequencies of C. mesnili ITS1 sequences differed significantly among years: the most abundant C. mesnili ITS1 sequence decreased and rare sequences increased over the course of the study, a pattern consistent with negative frequency-dependent selection. However, only a weak signal of host-genotype specificity between C. mesnili and Daphnia genotypes was detected. Use of cutting edge genomic techniques will allow further investigation of the underlying micro-evolutionary relationships within the Daphnia-C. mesnili system.

  9. Biochemical and growth acclimation of birch to night temperatures: genotypic similarities and differences.

    PubMed

    Mäenpää, M; Ossipov, V; Kontunen-Soppela, S; Keinänen, M; Rousi, M; Oksanen, E

    2013-01-01

    The responses of plants to environmental factors are connected to the time of day. In this study, silver birch (Betula pendula) was grown in growth chambers at five different night temperatures (6-22 °C), using gradual changes during the evening and morning hours. Despite the increased night respiration and unaffected daytime net photosynthesis (per square metre), the carbon uptake (biomass) of birch did not decrease, probably due to enhanced biochemical processes on warmer nights and the advantage of higher temperatures during the evening and morning hours. The plant stem height, internode length, stem dry weight (DW), stem mass fraction and specific leaf area increased with warmer night temperatures. Changes in growth and metabolite concentrations were partly nonlinear along the temperature gradient. Thus, the temperature effect depends on the temperature window considered. Genotypes had both common and genotype-specific biochemical responses to night temperatures. The common responses among genotypes were related to growth responses, whereas the unique responses may indicate genotype-specific differences in acclimation. The differences in genotypic growth and metabolite levels are valuable for assessing genotype qualities and understanding the connections between the metabolome and growth.

  10. Practical applications of genotypic surveys for forensic STR testing.

    PubMed

    Holt, C L; Stauffer, C; Wallin, J M; Lazaruk, K D; Nguyen, T; Budowle, B; Walsh, P S

    2000-08-14

    Legitimate genotype frequency estimation for multiallelic loci relies on component allele frequencies, as population surveys represent only a fraction of possible DNA profiles. Multilocus genotypes from two ethnic human populations, African American (n=195) and U.S. Caucasian (n=200), were compiled at 13 STR loci that are used worldwide in forensic investigation (D3S1358, vWA, FGA, D16S539, TH01, TPOX, CSF1PO, D8S1179, D21S11, D18S51, D5S818, D13S317, and D7S820). Sex-specific AmpFlSTR multiplexes provided stringent PCR-based STR typing specifically optimized for multicolor fluorescence detection. Heterozygosity at each STR locus ranged from 0.57 to 0.89 and encompassed from seven (TH01) to twenty-one (D21S11) alleles. Homozygosity tests, tests based on the distinct numbers of observed homozygous and heterozygous classes, log likelihood ratio tests, and exact tests assessed that the degree of divergence from theoretical Hardy-Weinberg proportions for all 13 STRs does not have practical consequence in genotype frequency estimation. Departures from linkage equilibrium, between loci, that imposed significance to forensic calculations were not indicated by observed variance of the number of heterozygous loci or Karlin interclass correlation tests. For forensic casework, reliable multilocus profile estimates may be obtained from the product of component genotype frequencies, each calculated through application of the Hardy-Weinberg equation to population database allele frequency estimates reported here. The average probability that two randomly selected, unrelated individuals possess an identical thirteen-locus DNA profile was one in 1.8x10(15) African Americans and one in 3.8x10(14) U.S. Caucasians. PMID:10940595

  11. Genotyping for cytochrome P450 polymorphisms.

    PubMed

    Daly, Ann K; King, Barry P; Leathart, Julian B S

    2006-01-01

    Protocols for the extraction of DNA from human blood and for genotyping for a number of common cytochrome P450 polymorphisms using either polymerase chain reaction (PCR)-restriction fragment length polymorphism or PCR-single-strand conformational polymorphism (SSCP) analysis are described. Rapid high-throughput techniques are also available for analyses of this type, but they require access to specialized equipment and are not considered here. General guidelines for performing amplification using PCR are described together with electrophoresis protocols for analysis of restriction digests of PCR products with agarose and polyacrylamide gels including the use of polyacrylamide-based gels for SSCP analysis. Protocols for the following specific isoforms and alleles are also provided: CYP1A1 (*2B and *4 alleles), CYP2C8 (*3 and *4 alleles), CYP2C9 (*2, *3, and *11 alleles), CYP2C19 (*2 and *3 alleles), CYP2D6 (*3, *4, *5, and *6 alleles), CYP2E1 (*5A, *5B, and *6 alleles), and CYP3A5 (*3 allele).

  12. Automated genotyping of circulating tumor cells.

    PubMed

    Stakenborg, Tim; Liu, Chengxun; Henry, Olivier; Borgen, Elin; Laddach, Nadja; Roeser, Tina; Ritzi-Lehnert, Marion; Fermér, Christian; Hauch, Sigfried; O'Sullivan, Ciara K; Lagae, Liesbet

    2010-09-01

    Cancer remains a prominent health concern in modern societies. Continuous innovations and introduction of new technologies are essential to level or reduce current healthcare spending. A diagnostic platform to detect circulating tumor cells (CTCs) in peripheral blood may be most promising in this respect. CTCs have been proposed as a minimally invasive, prognostic and predictive marker to reflect the biological characteristics of tumors and are implemented in an increasing number of clinical studies. Still, their detection remains a challenge as they may occur at concentrations below one single cell per ml of blood. To facilitate their detection, here we describe microfluidic modules to isolate and genotype CTCs directly from clinical blood samples. In a first cell isolation and detection module, the CTCs are immunomagnetically enriched, separated and counted. In a second module and after cell lysis, the mRNA is reversely transcripted to cDNA, followed by a multiplex ligation probe amplification of 20 specific genetic markers and two control fragments. Following the multiplex ligation probe amplification reaction, the amplified fragments are electrochemically detected in a third and final module. Besides the design of the modules, their functionality is described using control samples. Further testing using clinical samples and integration of all modules in a single, fully automated smart miniaturized system will enable minimal invasive testing for frequent detection and characterization of CTCs.

  13. Genotyping for cytochrome P450 polymorphisms.

    PubMed

    Daly, Ann K; King, Barry P; Leathart, Julian B S

    2006-01-01

    Protocols for the extraction of DNA from human blood and for genotyping for a number of common cytochrome P450 polymorphisms using either polymerase chain reaction (PCR)-restriction fragment length polymorphism or PCR-single-strand conformational polymorphism (SSCP) analysis are described. Rapid high-throughput techniques are also available for analyses of this type, but they require access to specialized equipment and are not considered here. General guidelines for performing amplification using PCR are described together with electrophoresis protocols for analysis of restriction digests of PCR products with agarose and polyacrylamide gels including the use of polyacrylamide-based gels for SSCP analysis. Protocols for the following specific isoforms and alleles are also provided: CYP1A1 (*2B and *4 alleles), CYP2C8 (*3 and *4 alleles), CYP2C9 (*2, *3, and *11 alleles), CYP2C19 (*2 and *3 alleles), CYP2D6 (*3, *4, *5, and *6 alleles), CYP2E1 (*5A, *5B, and *6 alleles), and CYP3A5 (*3 allele). PMID:16719392

  14. Flavonoid profile of green asparagus genotypes.

    PubMed

    Fuentes-Alventosa, J M; Jaramillo, S; Rodríguez-Gutiérrez, G; Cermeño, P; Espejo, J A; Jiménez-Araujo, A; Guillén-Bejarano, R; Fernández-Bolaños, J; Rodríguez-Arcos, R

    2008-08-27

    The determination of flavonoid profiles from different genotypes of triguero asparagus and their comparison to those from green asparagus commercial hybrids was the main goal of this study. The samples consisted of 32 commercial hybrids and 65 genotypes from the Huetor-Tajar population variety (triguero). The analysis of individual flavonoids by HPLC-DAD-MS has allowed the determination of eight naturally occurring flavonol derivatives in several genotypes of triguero asparagus. Those compounds included mono-, di-, and triglycosides of three flavonols, that is, quercetin, isorhamnetin, and kaempferol. The detailed analysis of the flavonoid profiles revealed significant differences among the distinct genotypes. These have been classified in three distinct groups as the result of a k-means clustering analysis, two of them containing both commercial hybrids and triguero asparagus and another cluster constituted by 21 genotypes of triguero asparagus, which contain several key flavonol derivatives able to differentiate them. Hence, the triglycosides tentatively identified as quercetin-3-rhamnosyl-rutinoside, isorhamnetin-3-rhamnosyl-rutinoside, and isorhamnetin-3-O-glucoside have been detected only in the genotypes grouped in the above-mentioned cluster. On the other hand, the compound tentatively identified as isorhamnetin-3-glucosyl-rutinoside was present in most genotypes of triguero asparagus, whereas it has not been detected in any of the commercial hybrids.

  15. Hepatitis C virus genotypes in Tirana, Albania.

    PubMed

    Haldeda, Migena; Baume, Julien; Tamalet, Catherine; Bizhga, Melpomeni; Colson, Philippe

    2014-01-01

    Hepatitis C virus (HCV) infection is a worldwide concern. Knowledge of the HCV genotype is clinically important because it predicts the rate of response to therapy and guides the treatment duration. Moreover, it allows molecular epidemiology to be performed. To our knowledge, the prevalence of HCV genotypes has been assessed only once in Albania, using a line probe genotyping assay. We determined HCV genotypes by population sequencing of HCV-infected patients in Tirana, Albania. HCV genotype and sequence analyses were performed for serum samples collected from January 2011 through May 2012 from 61 HCV-seropositive patients using population sequencing of the NS3 protease gene and alternatively the NS5b gene and the 5' untranslated region (UTR). HCV RNA was retrieved from the blood samples of 50 patients. The HCV NS3 protease gene was sequenced for 28 patients and NS5b and/or 5'UTR fragments were sequenced for an additional 22 patients. The predominant genotype was 1b in 25 patients (50%), followed by genotypes 2c, 4a, 3a, and 1a in 18%, 14%, 8%, and 6% of cases, respectively. Best matches for these HCV RNAs in GenBank were obtained in different countries worldwide. One NS3 protease naturally harbored an amino acid conferring minor drug resistance to newly available HCV protease inhibitors. In conclusion, HCV-1b was predominant in the present Albanian population, as in southeastern Europe.

  16. Distribution of Hepatitis C virus (HCV) genotypes in seropositive patients in the state of Alagoas, Brazil

    PubMed Central

    Gonzaga, Rosa Maria S.; Rodart, Itatiana F.; Reis, Mitermayer Galvão; Ramalho Neto, Cícero Eduardo; Silva, Denise Wanderlei

    2008-01-01

    We determined the frequency of hepatitis C virus (HCV) genotypes in anti-HCV seropositive patients in the state of Alagoas, Brazil, by means of nested-reverse transcription-polymerase chain reaction (RT-nested-PCR) followed by restriction fragment length polymorphism (RFLP) of amplified fragments of the 5´NCR. The nested-PCR with genotype-specific primers from the core region was carried out when detection was not possible by the first approach. Detectable HCV-RNA was present in 115 (74.7%) of 154 serum samples. Genotype 1 was the most frequent (77.4%), against 20.9% of genotype 3 and 0.8% of genotype 2. Subtype 1b was predominant (65.2%), followed by subtypes 1a (8.7%), and 3a (6.1%). Coinfection (1a/3a) was detected in 0.8% of the samples. Indeed, there was no significant differences in the prevalence of genotype 1 compared to what has been obtained from anti-HCV seropositive patients from other locations in Brazil. Here we report for the first time the genotype 2 in the state of Alagoas. PMID:24031281

  17. Host-parasite Red Queen dynamics with phase-locked rare genotypes.

    PubMed

    Rabajante, Jomar F; Tubay, Jerrold M; Ito, Hiromu; Uehara, Takashi; Kakishima, Satoshi; Morita, Satoru; Yoshimura, Jin; Ebert, Dieter

    2016-03-01

    Interactions between hosts and parasites have been hypothesized to cause winnerless coevolution, called Red Queen dynamics. The canonical Red Queen dynamics assume that all interacting genotypes of hosts and parasites undergo cyclic changes in abundance through negative frequency-dependent selection, which means that any genotype could become frequent at some stage. However, this prediction cannot explain why many rare genotypes stay rare in natural host-parasite systems. To investigate this, we build a mathematical model involving multihost and multiparasite genotypes. In a deterministic and controlled environment, Red Queen dynamics occur between two genotypes undergoing cyclic dominance changes, whereas the rest of the genotypes remain subordinate for long periods of time in phase-locked synchronized dynamics with low amplitude. However, introduction of stochastic noise in the model might allow the subordinate cyclic host and parasite types to replace dominant cyclic types as new players in the Red Queen dynamics. The factors that influence such evolutionary switching are interhost competition, specificity of parasitism, and degree of stochastic noise. Our model can explain, for the first time, the persistence of rare, hardly cycling genotypes in populations (for example, marine microbial communities) undergoing host-parasite coevolution.

  18. Screening selected genotypes of cowpea [Vigna unguiculata (L.) Walp.] for salt tolerance during seedling growth stage.

    PubMed

    Gogile, A; Andargie, M; Muthuswamy, M

    2013-07-15

    The environmental stress such as, salinity (soil or water) are serious obstacles for field crops especially in the arid and semi-arid parts of the world. This study was conducted to assess the potential for salt tolerance of cowpea genotypes during the seedling stage. The experimental treatments were 9 cowpea genotypes and 4 NaCl concentrations (0, 50, 100 and 200 mM) and they were tested in greenhouse. The experimental design was completely randomized design in factorial combination with three replications. Data analysis was carried out using SAS (version 9.1) statistical software. Seedling shoots and root traits, seedling shoots and root weight, number of leaves and total biological yield were evaluated. The analyzed data revealed highly significant (p < 0.001) variation among cowpea genotypes, treatments and their interactions. It is found that salt stress significantly decreased root length, shoot length, seedling shoot and root weight of cowpea genotypes. The extent of decrease varied with genotypes and salt concentrations. Most genotypes were highly susceptible to 200 mM NaCl concentration. The correlation analysis revealed positive and significant association among most of the parameters. Genotypes 210856, 211557 and Asebot were better salt tolerant. The study revealed the presence of broad intra specific genetic variation in cowpea varieties for salt stress with respect to their early biomass production.

  19. Prevalence and genotypes of Enterocytozoon bieneusi in sika deer in Jilin province, Northeastern China.

    PubMed

    Zhang, Xiao-Xuan; Cong, Wei; Liu, Guo-Hua; Ni, Xiao-Ting; Ma, Jian-Gang; Zheng, Wen-Bin; Zhao, Quan; Zhu, Xing-Quan

    2016-03-01

    Enterocytozoon bieneusi is one of the most important zoonotic pathogen that can infect almost all animals, including humans. However, little information is available regarding prevalence and genotypes of E. bieneusi in sika deer. In the present study, the prevalence of E. bieneusi infection in sika deer in Jilin province, Northeastern China was examined using PCR amplification of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene. 23 (7.06%) of 326 samples were tested E. bieneusi-positive, and the risk factor significantly associated with E. bieneusi prevalence was the age of sika deer. Sequence analysis of the ITS rRNA gene suggested that 8 genotypes of E. bieneusi were found in this study, with five known genotypes, namely J (n = 11), BEB6 (n = 4), EbpC (n = 1), CHN-DC1 (n = 1), KIN-1 (n = 1) and three novel genotypes, namely JLD-1 (n = 2), JLD-2 (n = 2) and JLD-3 (n = 1). Phylogenetic analysis indicated that genotypes CHN-DC-1, KIN-1, EbpC, JLD-2 and JLD-3 fell into group 1, while other three genotypes (genotypes J, BEB6 and JLD-1) were clustered into group 2 (so-called bovine-specific groups). These findings indicated the presence of zoonotic E. bieneusi in Jilin province, Northeastern China. Effective strategies should be performed to control E. bieneusi infection in sika deer, other animals and humans. PMID:27078663

  20. Host-parasite Red Queen dynamics with phase-locked rare genotypes

    PubMed Central

    Rabajante, Jomar F.; Tubay, Jerrold M.; Ito, Hiromu; Uehara, Takashi; Kakishima, Satoshi; Morita, Satoru; Yoshimura, Jin; Ebert, Dieter

    2016-01-01

    Interactions between hosts and parasites have been hypothesized to cause winnerless coevolution, called Red Queen dynamics. The canonical Red Queen dynamics assume that all interacting genotypes of hosts and parasites undergo cyclic changes in abundance through negative frequency-dependent selection, which means that any genotype could become frequent at some stage. However, this prediction cannot explain why many rare genotypes stay rare in natural host-parasite systems. To investigate this, we build a mathematical model involving multihost and multiparasite genotypes. In a deterministic and controlled environment, Red Queen dynamics occur between two genotypes undergoing cyclic dominance changes, whereas the rest of the genotypes remain subordinate for long periods of time in phase-locked synchronized dynamics with low amplitude. However, introduction of stochastic noise in the model might allow the subordinate cyclic host and parasite types to replace dominant cyclic types as new players in the Red Queen dynamics. The factors that influence such evolutionary switching are interhost competition, specificity of parasitism, and degree of stochastic noise. Our model can explain, for the first time, the persistence of rare, hardly cycling genotypes in populations (for example, marine microbial communities) undergoing host-parasite coevolution. PMID:26973878

  1. RT-PCR for detection of all seven genotypes of Lyssavirus genus.

    PubMed

    Vázquez-Morón, S; Avellón, A; Echevarría, J E

    2006-08-01

    The Lyssavirus genus includes seven species or genotypes named 1-7. Rabies genotypes correlate with geographical distribution and specific hosts. Co-circulation of different lyssaviruses, imported cases, and the presence of unknown viruses, such as Aravan, Khujand, Irkut and West Caucasian Bat Virus, make it necessary to use generic methods able to detect all lyssaviruses. Primer sequences were chosen from conserved regions in all genotypes in order to optimise a generic RT-PCR. Serial dilutions of 12 RNA extracts from all seven Lyssavirus genotypes were examined to compare the sensitivity of the RT-PCR standardised in this study with a published RT-PCR optimised for EBLV1 detection and capable of amplifying RNA from all seven lyssaviruses. All seven genotypes were detected by both RT-PCRs, however, the sensitivity was higher with the new version of the test. Twenty samples submitted for rabies diagnosis were tested by the new RT-PCR. Eight out of 20 samples from six dogs, one horse and one bat were found positive, in agreement with immunofluorescence results. Seven samples from terrestrial mammals were genotype 1 and one from a bat was genotype 5. In conclusion, this method can be used to complement immunofluorescence for the diagnosis of rabies, enabling the detection of unexpected lyssaviruses during rabies surveillance.

  2. ACTN3 genotype in professional soccer players.

    PubMed

    Santiago, C; González-Freire, M; Serratosa, L; Morate, F J; Meyer, T; Gómez-Gallego, F; Lucia, A

    2008-01-01

    The authors studied the frequency distribution of alpha-actinin-3 (ACTN3) R577X genotypes in 60 top-level professional soccer players. The results were compared with those of 52 elite endurance athletes and 123 sedentary controls. The per cent distribution of RR and RX genotypes in soccer players (48.3% and 36.7%) was significantly higher and lower, respectively, than controls (28.5% and 53.7%) and endurance athletes (26.5% and 52%) (p = 0.041). Although there are notable exceptions, elite soccer players tend to have the sprint/power ACTN3 genotype.

  3. Clinical significance of human papillomavirus genotyping.

    PubMed

    Choi, Youn Jin; Park, Jong Sup

    2016-03-01

    Cervical cancer is the fourth most common cancer in women worldwide, and the human papillomavirus (HPV) is the main causative agent for its development. HPV is a heterogeneous virus, and a persistent infection with a high-risk HPV contributes to the development of cancer. In recent decades, great advances have been made in understanding the molecular biology of HPV, and HPV's significance in cervical cancer prevention and management has received increased attention. In this review, we discuss the role of HPV genotyping in cervical cancer by addressing: clinically important issues in HPV virology; the current application of HPV genotyping in clinical medicine; and potential future uses for HPV genotyping.

  4. Molecular genotyping of human Ureaplasma species based on multiple-banded antigen (MBA) gene sequences.

    PubMed

    Kong, F; Ma, Z; James, G; Gordon, S; Gilbert, G L

    2000-09-01

    Ureaplasma urealyticum has been divided into 14 serovars. Recently, subdivision of U. urealyticum into two species has been proposed: U. parvum (previously U. urealyticum parvo biovar), comprising four serovars (1, 3, 6, 14) and U. urealyticum (previously U. urealyticum T-960 biovar), 10 serovars (2, 4, 5, 7-13). The multiple-banded antigen (MBA) genes of these species contain both species and serovar/subtype specific sequences. Based on whole sequences of the 5'-ends of MBA genes of U. parvum serovars and partial sequences of the 5'-ends of MBA genes of U. urealyticum serovars, we previously divided each of these species into three MBA genotypes. To further elucidate the relationships between serovars, we sequenced the whole 5'-ends of MBA genes of all 10 U. urealyticum serovars and partial repetitive regions of these genes from all serovars of U. parvum and U. urealyticum. For the first time, all four serovars of U. parvum were clearly differentiated from each other. In addition, the 10 serovars of U. urealyticum were divided into five MBA genotypes, as follows: MBA genotype A comprises serovars 2, 5, 8; MBA genotype B, serovar 10 only; MBA genotype C, serovars 4, 12, 13; MBA genotype D, serovar 9 only; and MBA genotype E comprises serovars 7 and 11. There were no sequence differences between members within each MBA genotype. Further work is required to identify other genes or other regions of the MBA genes that may be used to differentiate U. urealyticum serovars within MBA genotypes A, C and E. A better understanding of the molecular basis of serotype differentiation will help to improve subtyping methods for use in studies of the pathogenesis and epidemiology of these organisms.

  5. High-throughput genotyping: practical considerations concerning the day-to-day application

    NASA Astrophysics Data System (ADS)

    McIndoe, Richard A.; Bumgarner, R. E.; Welti, Russ; Hood, Leroy

    1996-04-01

    Advances in high throughput genotyping protocols over the past few years have been remarkable. Most protocols developed to increase the throughput of genotyping rely on fluorescent based technologies for data acquisition and capture. In general, the number of genotypes per day quoted for these protocols are the result of extrapolations based on ideal situations. Here we present our experience with respect to the day to day problems of high throughput genotyping. Our laboratory is currently working on several genetic mapping projects in both mouse and man. For example, we are looking at the genetic basis for susceptibility to rheumatoid arthritis in a local native American tribe as well as a mouse animal model for the same disease. The machines used to collect gel image data are two Li- Cor infrared DNA sequencers adapted for genotyping. During the evolution of these projects, we have addressed issues concerning the tracking and flow of information from the initial extraction of DNA to the calling of the genotypes. In particular, we have focused on designing methods that are efficient, cost effective and can be easily taught to the technical staff. Computer programs have been written that record gel specific information (e.g. ID information), archive data and capture genotypes in a simple point and click environment. Instrumentation was purchased to ease the repetitive nature of sample allocation, reagent disbursement and gel loading. Using this system, we can produce genotype data on 96 individuals for 20 loci (1920 genotypes) in one day. Solutions to the overall flow of information at each of these junctions are discussed.

  6. Temporal dynamics and decay of putatively allochthonous and autochthonous viral genotypes in contrasting freshwater lakes.

    PubMed

    Hewson, Ian; Barbosa, Jorge G; Brown, Julia M; Donelan, Ryan P; Eaglesham, James B; Eggleston, Erin M; LaBarre, Brenna A

    2012-09-01

    Aquatic viruses play important roles in the biogeochemistry and ecology of lacustrine ecosystems; however, their composition, dynamics, and interactions with viruses of terrestrial origin are less extensively studied. We used a viral shotgun metagenomic approach to elucidate candidate autochthonous (i.e., produced within the lake) and allochthonous (i.e., washed in from other habitats) viral genotypes for a comparative study of their dynamics in lake waters. Based on shotgun metagenomes prepared from catchment soil and freshwater samples from two contrasting lakes (Cayuga Lake and Fayetteville Green Lake), we selected two putatively autochthonous viral genotypes (phycodnaviruses likely infecting algae and cyanomyoviruses likely infecting picocyanobacteria) and two putatively allochthonous viral genotypes (geminiviruses likely infecting terrestrial plants and circoviruses infecting unknown hosts but common in soil libraries) for analysis by genotype-specific quantitative PCR (TaqMan) applied to DNAs from viruses in the viral size fraction of lake plankton, i.e., 0.2 μm > virus > 0.02 μm. The abundance of autochthonous genotypes largely reflected expected host abundance, while the abundance of allochthonous genotypes corresponded with rainfall and storm events in the respective catchments, suggesting that viruses with these genotypes may have been transported to the lake in runoff. The decay rates of allochthonous and autochthonous genotypes, assessed in incubations where all potential hosts were killed, were generally lower (0.13 to 1.50% h(-1)) than those reported for marine virioplankton but similar to those for freshwater virioplankton. Both allochthonous and autochthonous viral genotypes were detected at higher concentrations in subsurface sediments than at the water-sediment interface. Our data indicate that putatively allochthonous viruses are present in lake plankton and sediments, where their temporal dynamics reflect active transport to the lake during

  7. Serological Assay and Genotyping of Hepatitis C Virus in Infected Patients in Zanjan Province

    PubMed Central

    Esmaeilzadeh, Abdolreza; Erfanmanesh, Maryam; Ghasemi, Sousan; Mohammadi, Farzaneh

    2014-01-01

    Background: Hepatitis C Virus (HCV), a public health problem, is an enveloped, single-stranded RNA virus and a member of the Hepacivirus genus of the Flaviviridae family. Liver cancer, cirrhosis, and end-stage liver are the outcomes of chronic infection with HCV. HCV isolates show significant heterogeneity in genetics around the world. Therefore, determining HCV genotypes is a vital step in determining prognosis and planning therapeutic strategies. Objectives: As distribution of HCV genotypes is different in various geographical regions and HCV genotyping of patients has not been investigated in Zanjan City, this study was designed for the first time, to determine HCV genotypes in the region and to promote the impact of the treatment. Materials and Methods: Serum samples of 136 patients were collected and analyzed for anti-HCV antibodies using ELISA (The enzyme-linked immunosorbent assay) method. Then, positive samples were exposed to RT-PCR, which was performed under standard condition. Afterwards, they investigated for genotyping using allele-specific PCR (AS-PCR), and HCV genotype 2.0 line probe assay (LiPA). Results: Samples indicated 216 bp bands on 2% agarose gel. Analyses of the results demonstrated that the most dominant subtype was 3a with frequency of 38.26% in Zanjan Province followed by subtypes of 1b, 1a, 2, and 4 with frequencies of 25.73%, 22.05%, 5.14%, and 4.41%, respectively. The frequency of unknown HCV genotypes was 4.41%. Conclusions: According to the results, it was found that HCV high prevalent genotype in Zanjan is subtype 3a. Analysis of the results provides identification of certain HCV genotypes, and these valuable findings could affect the type and duration of the treatment. PMID:25368655

  8. Breakpoint analysis: Precise localization of genetic markers by means of nonstatistical computation using relatively few genotypes

    SciTech Connect

    Elsner, T.I.; Albertsen, H.; Gerken, S.C.; Cartwright, P.; White, R.

    1995-02-01

    Placing new markers on a previously existing genetic map by using conventional methods of multilocus linkage analysis requires that a large number of reference families be genotyped. This paper presents a methodology for placing new markers on existing genetic maps by genotyping only a few individuals in a selected subset of the reference panel. We show that by identifying meiotic breakpoint events within existing genetic maps and genotyping individuals who exhibit these events, along with one nonrecombinant sibling and their parents, we can determine precise locations for new markers even within subcentimorgan chromosomal regions. This method also improves detection of errors in genotyping and assists in the observation of chromosome behavior in specific regions. 31 refs., 9 figs.

  9. High Diversity of Hepatitis B Virus Genotypes in Panamanian Blood Donors: A Molecular Analysis of New Variants

    PubMed Central

    Martínez, Alexander A.; Zaldivar, Yamitzel Y.; De Castillo, Zoila; Ortiz, Alma Y.; Mendoza, Yaxelis; Cristina, Juan; Pascale, Juan M.

    2014-01-01

    Hepatitis B Virus (HBV) is an infectious agent that causes more than half of the cases of liver disease and cancer in the world. Globally there are around 250 million people chronically infected with this virus. Despite 16% of the cases of liver disease in Central America are caused by HBV, the information regarding its genetic diversity, genotypes and circulation is scarce. The purpose of this study was to evaluate the genetic variability of the HBV genotypes from HBV-DNA positive samples obtained from screening blood donors at the Social Security System of Panama and to estimate its possible origin. From 59,696 blood donors tested for HBV infection during 2010–2012, there were 74 HBV-DNA positive subjects. Analysis of the partial PreS2-S region of 27 sequences shows that 21% of the infections were caused by genotype A, 3% by genotype D and 76% by genotype F. In addition, we were able to confirm circulation of six sub-genotypes A1, A2, A3, D4, F3, F1 and a proposed new sub-genotype denominated F5pan. We found a confinement of sub-genotypes F1 and F5pan to the western area of Panama.