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Sample records for b19 genotype specific

  1. Parvovirus B19-specific DNA in bone marrow from B19 arthropathy patients: evidence for B19 virus persistence.

    PubMed

    Foto, F; Saag, K G; Scharosch, L L; Howard, E J; Naides, S J

    1993-03-01

    To determine if B19 infection persists in patients with chronic B19 arthropathy, acute B19 infection in adults was documented by IgM serology. Bone marrow aspirates were obtained 24-42 months after acute infection in 4 patients who developed chronic joint symptoms. DNA from bone marrow aspirates was amplified by polymerase chain reaction by using B19-specific DNA sequences in the viral capsid gene and probing with B19-specific oligonucleotides in Southern analysis. B19-specific DNA sequences were detected in all 4 chronic arthropathy patients compared with 0 of 7 anti-B19 IgM-, IgG- and 0 of 6 anti-B19 IgM-, IgG+ normal bone marrow donors. Three patients with serologically proven acute B19 infection and acute but nonchronic joint symptoms had B19 DNA detected in bone marrow aspirates 2-18 months after infection. These findings suggest that B19 arthropathy is associated with persistence of either B19 virus or select B19 DNA sequences. PMID:8440942

  2. Prevalence and genotypic characterization of Human Parvovirus B19 in children with measles- and rubella-like illness in Iran.

    PubMed

    Rezaei, Farhad; Sarshari, Behrang; Ghavami, Nastaran; Meysami, Parisa; Shadab, Azadeh; Salimi, Hamid; Mokhtari-Azad, Talat

    2016-06-01

    Human Parvovirus B19 (B19V) is a prototype of the Erythroparvovirus genus in Parvoviridae family. B19V infections are often associated with fever and rash, and can be mistakenly reported as measles or rubella. Differential diagnosis of B19V illness is necessary for case management and also for public health control activities, particularly in outbreak situations in which measles or rubella is suspected. To investigate the causative role of B19V infection in children with measles- and rubella-like illness, a total of 583 sera from children with exanthema were tested for presence of B19V by determining anti-B19V IgG and IgM antibodies by ELISA as well as B19V DNA detection by nested PCR. DNA positive samples were assessed further for determination of viral load and sequence analysis by Real-Time PCR and Sanger sequencing method, respectively. Out of 583 patients, 112 (19.21%) patients were positive for B19V-IgM antibody, 110 (18.87%) were positive for B19V-IgG antibody, and 63 (10.81%) were positive for B19V viral DNA. The frequency of B19V-IgG antibodies were increased with age; that is children under 6 year old showed 7.11% seroprevalence for B19V-IgG as compared to 18.39% and 28.91% for age groups 6 to >11 and 11-14 years old, respectively. Phylogenetic analysis of the NS1-VPu1 overlapping region revealed that all sequenced B19V-DNA belonged to genotype 1. The results of this study may aid the surveillance programs aiming at eradicating measles/rubella virus in Iran, as infections with B19V can be mistakenly reported as measles or rubella if laboratory testing is not conducted. PMID:26538067

  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. Prolonged Activation of Virus-Specific CD8+T Cells after Acute B19 Infection

    PubMed Central

    2005-01-01

    Background Human parvovirus B19 (B19) is a ubiquitous and clinically significant pathogen, causing erythema infectiosum, arthropathy, transient aplastic crisis, and intrauterine fetal death. The phenotype of CD8+ T cells in acute B19 infection has not been studied previously. Methods and Findings The number and phenotype of B19-specific CD8+ T cell responses during and after acute adult infection was studied using HLA–peptide multimeric complexes. Surprisingly, these responses increased in magnitude over the first year post-infection despite resolution of clinical symptoms and control of viraemia, with T cell populations specific for individual epitopes comprising up to 4% of CD8+ T cells. B19-specific T cells developed and maintained an activated CD38+ phenotype, with strong expression of perforin and CD57 and downregulation of CD28 and CD27. These cells possessed strong effector function and intact proliferative capacity. Individuals tested many years after infection exhibited lower frequencies of B19-specific cytotoxic T lymphocytes, typically 0.05%–0.5% of CD8+ T cells, which were perforin, CD38, and CCR7 low. Conclusion This is the first example to our knowledge of an “acute” human viral infection inducing a persistent activated CD8+ T cell response. The likely explanation—analogous to that for cytomegalovirus infection—is that this persistent response is due to low-level antigen exposure. CD8+ T cells may contribute to the long-term control of this significant pathogen and should be considered during vaccine development. PMID:16253012

  5. 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

  6. 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 ...

  7. [Genotypic specificity of schizophrenic psychoses].

    PubMed

    Shakhmatova-Pavlova, I V; Gindilis, V M; Rokhlina, M L; Kozlova, I A

    1980-01-01

    A total of 610 probands with a disease manifestation in childhood, middle and old age and their families were examined by the clinico-genealogical method. The results allowed conclusions that (1) there is an undoubted genetic relationship between schizophrenia of childhood, middle and old age; and that (2) among the closest relatives in families of the probands there is no significant (in comparison to the general population) accumulation of non-schizophrenic pathology. The latter indicates a high genotypic specificity of schizophrenia. PMID:7415694

  8. Parvovirus B19.

    PubMed

    Landry, Marie Louise

    2016-06-01

    Primary parvovirus B19 infection is an infrequent, but serious and treatable, cause of chronic anemia in immunocompromised hosts. Many compromised hosts have preexisting antibody to B19 and are not at risk. However, upon primary infection, some patients may be able to mount a sufficient immune response to terminate active parvovirus B19 infection of erythroid precursors. The most common consequence of B19 infection in the compromised host is pure red-cell aplasia, resulting in chronic or recurrent anemia with reticulocytopenia. Anemia persists until neutralizing antibody is either produced by the host or passively administered. Parvovirus B19 should be suspected in compromised hosts with unexplained or severe anemia and reticulocytopenia, or when bone-marrow examination shows either giant pronormoblasts or absence of red-cell precursors. Diagnosis is established by detection of B19 DNA in serum in the absence of IgG antibody to B19. In some cases, IgG antibody is detected but is not neutralizing. Anti-B19 IgM may or may not be present. Therapy includes any or all of the following: red-cell transfusion, adjustment in medications to restore or improve the patient's immune system, and administration of intravenous immunoglobulin (IVIG). Following treatment, patients should be closely monitored, especially if immunosuppression is unchanged or increased. Should hematocrit trend downward and parvovirus DNA trend upward, the therapeutic options above should be revisited. In a few instances, monthly maintenance IVIG may be indicated. Caregivers should be aware that B19 variants, though rarely encountered, can be missed or under-quantitated by some real-time polymerase-chain reaction methods. PMID:27337440

  9. 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...

  10. 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

  11. 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

  12. Parvovirus B19 and Other Illnesses

    MedlinePlus

    ... Cheek Rash Parvovirus B19 and Other Illnesses References Parvovirus B19 and Other Illnesses Recommend on Facebook Tweet Share ... disease is the most common illness caused by parvovirus B19 infection. Learn More Parvovirus B19 infection can cause ...

  13. 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

  14. Seroprevalence of erythrovirus B19 in Saudi pregnant women

    PubMed Central

    Johargy, Ayman K.

    2016-01-01

    Background: Erythrovirus B19 infection is associated with clinical symptoms that range from mild to severe. The common clinical presentation of B19 virus (B19V) infection is erythema infectiosum, arthropathy, aplastic crisis, and fetal infection. Infection in seronegative pregnant women can lead to fetal hydrops. Objectives: To determine the seroprevalence of immunoglobulin G (IgG) to erythrovirus B19 in Saudi pregnant women in the cities of Makkah and Jeddah in Saudi Arabia. Materials and Methods: A total of 364 blood (serum) samples were tested for erythrovirus B19-specific-IgG antibody in Saudi pregnant women in the cities of Makkah and Jeddah in Saudi Arabia. Results: Erythrovirus B19-specific-IgG antibodies were detected in 182/364 (50%) of Saudi pregnant women of different age groups. Conclusion: This study indicated that B19V is clearly circulating in the community in a way that is similar to what is found in most nontemperate countries. PMID:27186157

  15. 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...

  16. 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...

  17. 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...

  18. 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...

  19. 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. PMID:26756922

  20. Fifth Disease (Parvovirus B19) and Pregnancy

    MedlinePlus

    Fifth Disease (parvovirus B19) In every pregnancy, a woman starts out with a 3-5% chance of having a baby with a ... infectiosum, is a viral illness caused by human parvovirus B19. It occurs most commonly in children ages 4 ...

  1. 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

  2. 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

  3. Detection of antibodies and antigens of human parvovirus B19 by enzyme-linked immunosorbent assay.

    PubMed Central

    Anderson, L J; Tsou, C; Parker, R A; Chorba, T L; Wulff, H; Tattersall, P; Mortimer, P P

    1986-01-01

    Acute-phase serum from a patient with aplastic crisis provided sufficient human parvovirus B19 to make a monoclonal antibody against B19 and to develop antigen and immunoglobulin M (IgM) and IgG antibody detection enzyme-linked immunosorbent assays (ELISAs). The indirect capture antibody method was used for all three assays. Antigen was detected in 8 of 29 sera drawn within 2 days of onset of illness from patients with aplastic crisis. These sera had high titers of virus by electron microscopy and DNA hybridization and had no detectable B19 antibody. Antigen was not detected in serum specimens that had low titers of B19 DNA and had B19 antibody. With the IgM ELISA, we detected B19 IgM in over 85% of clinical cases of aplastic crisis and fifth disease and less than 2% of controls. The prevalence of B19 IgG antibodies increased with age. Approximately 2% of children less than 5 years of age and 49% of adults greater than 20 years of age had B19 IgG antibodies. The B19 antibody ELISAs are sensitive and specific tests to detect B19 infections. PMID:3021807

  4. Detection of Parvovirus B19 Infection in Thalasemic Patients in Isfahan Province, Iran

    PubMed Central

    Nikoozad, Razieh; Mahzounieh, Mohammad Reza; Ghorani, Mohammad Reza

    2015-01-01

    Background: Parvovirus B19, a member of the Erythrovirus genus of Parvoviridae family, causes various clinical illnesses including infectious erythema, arthropathy, hydrops fetalis or congenital anemia, and transient aplastic crises. The B19 virus can be transmitted through respiratory secretions, blood products, and blood transfusion. Objectives: The aim of this study was to detect the B19 virus in thalassemia patients in Isfahan, Iran. Patients and Methods: The prevalence of parvovirus B19 infection was compared between thalassemia major patients and healthy subjects. Plasma samples were collected from 30 thalassemia patients from Isfahan, Iran. Thirty patients without any blood complications were considered as the control group. After DNA extraction from the plasma samples, polymerase chain reaction was performed for parvovirus B19 detection. Results: The parvovirus B19-specific nucleotide sequence was detected in 6 patients (20%). None of the samples obtained from the 30 control subjects tested positive for B19. Conclusions: In this study B19-Parvovirus infection were detected in patients with hematologic disorders in comparison with control subjects. Screening of patients with a high risk of parvovirus B19 infection can considerably reduce the incidence and prevalence of B19 infection. PMID:26855745

  5. 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. PMID:16998895

  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. No evidence of persistent parvovirus B19 viremia among Iranian patients with HIV after a 1-year follow-up.

    PubMed

    Aghakhani, Arezoo; Mohraz, Minoo; Azadmanesh, Kayhan; Moayedi-Nia, Saeedeh; Kazemimanesh, Monireh; Mamishi, Setareh; Banifazl, Mohammad; Ramezani, Amitis

    2016-05-01

    Recent studies have demonstrated that, in common with other latent viruses, parvovirus B19 infection can be controlled by the host immune response but may persist in some places such as the bone marrow. Persistent B19 infection has been found in both immunocompetent and immunocompromised individuals, such as patients infected with human immunodeficiency virus (HIV). However, there is limited data regarding long-term B19 viremia in HIV patients. In this study, we investigated virological and hematological findings, and also the clinical outcome, of seven cases of HIV/B19 coinfection (confirmed by PCR) after one year. These cases were provided from a previous study on patients with HIV infection that found B19 DNA in 13 cases. Seven of these 13 patients were available after 1 year, and we retested them for B19 viremia and B19-specific antibodies. B19 IgG was tested by ELISA, and B19 DNA was assessed by nested PCR. Anemia was not observed in these cases. All subjects had cleared viremia, but B19 IgG seroconversion occurred in two cases. No significant changes in CD4 and hemoglobin occurred. The results of this study indicate that B19 infection in HIV patients is a subtle infection and that B19 viremia is not a long-term event. PMID:26860911

  8. Enhanced inhibition of parvovirus B19 replication by cidofovir in extendedly exposed erythroid progenitor cells.

    PubMed

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

    2016-07-15

    Human parvovirus B19 (B19V) commonly induces self-limiting infections but can also cause severe clinical manifestations in patients with underlying haematological disorders or with immune system deficits. Currently, therapeutic options for B19V entirely rely on symptomatic and supportive treatments since a specific antiviral therapy is not yet available. Recently a first step in the research for active compounds inhibiting B19V replication has allowed identifying the acyclic nucleoside phosphonate cidofovir (CDV). Herein, the effect of CDV against B19V replication was characterized in human erythroid progenitor cells (EPCs) cultured and infected following different experimental approaches to replicate in vitro the infection of an expanding erythroid cell population in the bone marrow. B19V replication was selectively inhibited both in infected EPCs extendedly exposed to CDV 500μM (viral inhibition 82%) and in serially infected EPCs cultures with passage of the virus progeny, constantly under drug exposure (viral inhibition 99%). In addition, a potent inhibitory effect against B19V (viral inhibition 92%) was assessed in a short-term infection of EPCs treated with CDV 500μM 1day before viral infection. In the evaluated experimental conditions, the enhanced effect of CDV against B19V might be ascribed both to the increased intracellular drug concentration achieved by extended exposure, and to a progressive reduction in efficiency of the replicative process within treated EPCs population. PMID:27071853

  9. 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. PMID:22499021

  10. 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

  11. Human parvovirus B19: relevance in internal medicine.

    PubMed

    van Elsacker-Niele, A M; Kroes, A C

    1999-06-01

    Infection by the human parvovirus B19 can lead to several clinical manifestations which are relevant in internal medicine. These include aplastic crisis in chronic haemolytic anaemias, exanthemathous disease and arthropathy, mainly in women, and chronic anaemia in the immunocompromised host. After initial replication, probably in the respiratory tract, the virus enters its target cells in the bone marrow, erythroid precursor cells, through its receptor, the blood group P antigen. Viral replication in these cells leads to an arrest in erythropoiesis, normally lasting approximately 1 week. In this stage, an aplastic crisis can be produced in all patients under 'erythropoietic stress'. The viraemia disappears as specific antibodies to the virus become detectable in serum, which may give rise to a rash or arthralgia, symptoms that are probably immune-mediated. In immunologically normal individuals the infection is cleared by the humoral immune system within several weeks, whereafter detectable specific IgG confers lifelong immunity to reinfection. In patients with absent or dysfunctional humoral immunity to this virus, however, persistent infection can occur, which results in chronic suppression of erythropoiesis with chronic anaemia. Passive immunization, by means of normal immunoglobulin preparations has been reported to be effective in treating this condition. Diagnosis of parvovirus infection is usually possible by the detection of specific antibodies of IgM class in cases of recent infection. In patients with aplastic crisis and patients with chronic anaemia diagnosis rests upon the detection of parvovirus B19 DNA in serum by polymerase chain reaction. Parvovirus B19 is a ubiquitous virus. By the age of 15, about 50% of individuals have serologic evidence of a past infection, which may present as the common childhood disease erythema infectiosum. At the age of 70, seroprevalence reaches 80 to 100%. A vaccine against this virus is currently being developed. PMID

  12. 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

  13. Prolonged remission in a child with chronic myeloid leukemia following Parvo virus B19 (B19V) infection.

    PubMed

    Kumar, A; Moulik, N Roy; Kishore, J; Kumar, A; Jain, A

    2015-01-01

    Parvovirus B19 (B19V) has been associated with a wide spectrum of clinico-pathological disorders in human beings depending upon the host immunity. The present report describes a child with chronic myeloid leukemia ( CML) on hydroxyurea in haematological remission, who developed profound erythroid suppression following B19V infection requiring multiple transfusions and withdrawal of hydroxyurea. Despite being off-therapy the child remained in complete clinical and haematological remission till anti B19V antibodies appeared. This case illustrates the ability of B19V infection in suppressing neoplastic myeloid clone, a phenomenon not described earlier. PMID:26068352

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

    PubMed

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

    2016-04-01

    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 HbSβ(0)-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

  15. 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

  16. 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

  17. 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

  18. Low host-specific Enterocytozoon bieneusi genotype BEB6 is common in Swedish lambs.

    PubMed

    Stensvold, Christen Rune; Beser, Jessica; Ljungström, Bitte; Troell, Karin; Lebbad, Marianne

    2014-09-15

    Enterocytozoon bieneusi was found in 49/72 (68%) fecal samples from Swedish lambs while 37 samples from 24 adult sheep were negative. Molecular characterization of the internal transcribed spacer (ITS) revealed three genotypes: BEB6, OEB1, and OEB2, the latter two of which were novel and all three of which belonged to a group of genotypes (Group 2) of probably limited zoonotic importance. Our observations suggest that E. bieneusi is common in young Swedish sheep and add support to emerging evidence that the common BEB6 genotype, originally thought to be adapted to cattle, is capable of infecting a variety of hosts, suggesting low host specificity. PMID:24986434

  19. Manganese deficiency leads to genotype-specific changes in fluorescence induction kinetics and state transitions.

    PubMed

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

    2009-06-01

    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

  20. Characteristic genotypes discriminate between Babesia canis isolates of differing vector specificity and pathogenicity to dogs.

    PubMed

    Zahler, M; Schein, E; Rinder, H; Gothe, R

    1998-07-01

    The first and second internal transcribed spacers (ITS1, ITS2) as well as the intervening 5.8S coding region of the rRNA gene were characterized in eight Babesia canis isolates of differing geographic origin, vector specificity, and pathogenicity to dogs. The genotypes determined by sequencing segregated into three clearly separated groups close to or near the species level and correspond to the previously proposed subspecies B. canis canis, B. canis vogeli, and B. canis rossi. The three genotypes can be distinguished by Sau96I digestion of the polymerase chain reaction (PCR)-amplified rDNA target. PMID:9694369

  1. 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

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

    PubMed

    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

  3. 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. PMID:26927158

  4. 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

  5. Prevalence and Viral Load of Human Parvovirus B19 (B19V) Among Blood Donors in South-East Brazil.

    PubMed

    Slavov, Svetoslav Nanev; Otaguiri, Katia Kaori; Covas, Dimas Tadeu; Kashima, Simone

    2016-06-01

    The infection of human parvovirus B19 (B19V) is a common event in the general population, including volunteer blood donors. In some cases it can be asymptomatic and can remain persistent for a long period of time. The objective of this study was to examine the B19V DNA prevalence and viral load in first-time volunteer blood donors. Blood samples were collected from 91 primary blood donors at the Regional Blood Center of Ribeirão Preto, Southeast Brazil. Viral detection and quantitation was performed by an in-house TaqMan(®) real-time PCR with high sensitivity. B19V DNA was detected in one male blood donor (1.0 %) and was characterized by a very low viral load (537.36 copies/mL). Our studies demonstrate that B19V DNA at low titer may be present in apparently healthy individuals. Sensitive molecular diagnostic tools can be applied for the screening of fresh blood derived products in order to prevent transfusion-transmitted B19V infection. PMID:27408426

  6. Specific Polymorphisms in Hepatitis C Virus Genotype 3 Core Protein Associated with Intracellular Lipid Accumulation

    PubMed Central

    Jhaveri, Ravi; McHutchison, John; Patel, Keyur; Qiang, Guan; Diehl, Anna Mae

    2008-01-01

    Background Steatosis is a common histological finding and a poor prognostic indicator in patients with hepatitis C virus (HCV) infection. In HCV genotype 3–infected patients, the etiology of steatosis appears to be closely correlated with unknown viral factors that increase intracellular lipid levels. We hypothesize that specific sequence polymorphisms in HCV genotype 3 core protein may be associated with hepatic intracellular lipid accumulation. Methods Using selected serum samples from 8 HCV genotype 3–infected patients with or without steatosis, we sequenced the HCV core gene to identify candidate polymorphisms associated with increased intracellular lipid levels. Results Two polymorphisms at positions 182 and 186 of the core protein correlated with the presence (P = .03) and absence (P = .005) of intrahepatic steatosis. Transfected liver cell lines expressing core protein with steatosis-associated polymorphisms had increased intracellular lipid levels compared with non–steatosis-associated core isolates, as measured by oil red O staining (P = .02). Site-specific mutagenesis performed at positions 182 and 186 in steatosis-associated core genes yielded proteins that had decreased intracellular lipid levels in transfected cells (P = .03). Conclusions We have identified polymorphisms in HCV core protein genotype 3 that produce increased intracellular lipid levels and thus may play a significant role in lipid metabolism or trafficking, contributing to steatosis. PMID:18177246

  7. Parvovirus B19V infection in Israel: prevalence and occurrence of acute infection between 2008 and 2013.

    PubMed

    Mor, O; Ofir, I; Pavel, R; Bassal, R; Kra-Oz, Z; Cohen, D; Shohat, T; Mendelson, E

    2016-01-01

    Differences in the seroprevalence and unique pattern of parvovirus B19 (B19V) acute infections have been documented around the world. This study was conducted to estimate the seroprevalence of anti-parvovirus B19V IgG antibodies in the Israeli population and to assess the pattern of acute infection based on data from two laboratories in Israel. The overall IgG prevalence in the 1008 representative sera samples was 61·4% and the age-adjusted prevalence rate was 58·2%. Seropositivity was significantly associated with age, ranging from 25·7% in children aged 20 years. While no significant differences in seropositivity were detected between sexes and population groups, significantly lower seroprevalence was observed in older Jews born in Africa or Asia. Acute infection rates of 4·1% (234 cases) were found based on the positive IgM results identified in samples from 5663 individuals collected between 2008 and 2013. Annual peaks of infection were observed in 2008 and 2011-2012 and major seasonal peak of B19V IgM positivity was identified in June each year. The number of requests for B19V serology was significantly higher for women aged 20-39 years while the majority IgM-positive cases were identified in young children. With more than 30% of the adult population being susceptible to B19V infection, monitoring B19V status should be considered in specific risk groups such as pregnant women. PMID:25990962

  8. MICB Allele Genotyping on Microarrays by Improving the Specificity of Extension Primers

    PubMed Central

    Baek, In-Cheol; Jang, Jung-Pil; Choi, Eun-Jeong; Kim, Tai-Gyu

    2015-01-01

    Major histocompatibility complex (MHC) class I chain-related gene B (MICB) encodes a ligand for activating NKG2D that expressed in natural killer cells, γδ T cells, and αβ CD8+ T cells, which is associated with autoimmune diseases, cancer, and infectious diseases. Here, we have established a system for genotyping MICB alleles using allele-specific primer extension (ASPE) on microarrays. Thirty-six high quality, allele-specific extension primers were evaluated using strict and reliable cut-off values using mean fluorescence intensity (MFI), whereby an MFI >30,000 represented a positive signal and an MFI <10,000 represented a negative signal. Eight allele-specific extension primers were found to be false positives, five of which were improved by adjusting their length, and three of which were optimized by refractory modification. The MICB alleles (*002:01, *003, *005:02/*010, *005:03, *008, *009N, *018, and *024) present in the quality control panel could be exactly defined by 22 allele-specific extension primers. MICB genotypes that were identified by ASPE on microarrays were in full concordance with those identified by PCR-sequence-based typing. In conclusion, we have developed a method for genotyping MICB alleles using ASPE on microarrays; which can be applicable for large-scale single nucleotide polymorphism typing studies of population and disease associations. PMID:26569110

  9. Detection of parvovirus B19 in donated blood: a model system for screening by polymerase chain reaction.

    PubMed Central

    McOmish, F; Yap, P L; Jordan, A; Hart, H; Cohen, B J; Simmonds, P

    1993-01-01

    A highly sensitive and rapid method for routinely screening large numbers of donated blood units for parvovirus B19 by the polymerase chain reaction (PCR) was developed. Over a 3-month trial period in Edinburgh, B19 DNA was detected in 6 of 20,000 consecutive units of blood (0.03%), in concentrations ranging from 2.4 x 10(4) to 5 x 10(10) copies of viral DNA per ml. Seroconversion for B19-specific immunoglobulin M and immunoglobulin G and disappearance of circulating B19 DNA occurred in the interval between donation and recall in four of the five implicated donors who could be recalled. B19 DNA was detected in 18 of 27 separate batches of non-heat-treated factor VIII and IX concentrate manufactured from donated plasma unscreened for B19 DNA. Dry-heat treatment at 80 degrees C for 72 h reduced but did not always eliminate detectable B19 from factor VIII concentrates, consistent with recent observations that current methods for virus inactivation during blood product manufacture are insufficient to entirely eliminate B19 infectivity. The methods developed in this study for PCR screening could be applied routinely to prevent transfusion of B19 in blood and blood products and could play an important role in the prevention of iatrogenic transmission of infection. PCR screening could also be used for detection and exclusion of a range of other transmission-associated viruses for which current serological detection methods are only partially effective. PMID:8432819

  10. Quantitative genotyping of single-nucleotide polymorphisms by allele-specific oligonucleotide hybridization on DNA microarrays.

    PubMed

    Rickert, Andreas M; Ballvora, Agim; Matzner, Ulrich; Klemm, Manfred; Gebhardt, Christiane

    2005-08-01

    Genotyping of SNPs (single-nucleotide polymorphisms) has challenged the development of several novel techniques. Most of these methods have been introduced to discriminate binary SNPs in diploid species. In the present study, the quantitative genotyping of SNPs in natural DNA pools of a polyploid organism via DNA microarrays was analysed. Three randomly selected SNP loci were genotyped in the tetraploid species potato (Solanum tuberosum). For each SNP, 24 oligomers were designed, 12 with forward and 12 with reverse orientation. They contained the polymorphic site at one of the positions 11, 14 and 17. Several steps of optimizations were performed, including the 'materials' used and the establishment of hybridization conditions. Glass surfaces were either epoxy- or aldehyde-modified, and allele-specific oligonucleotides contained either SH or NH2 groups. Hybridization stringency conditions were established by varying the concentration of formamide in the hybridization buffer. For SNP BA213c14t7/403, the quantitative discrimination between all four different naturally occurring genotypes could be demonstrated. PMID:15847606

  11. The cutaneous manifestations of human parvovirus B19 infection.

    PubMed

    Magro, C M; Dawood, M R; Crowson, A N

    2000-04-01

    The prototypical cutaneous manifestations of human parvovirus B19 (B19) infection include a petechial eruption in a glove and stocking distribution, reticular truncal erythema, and the "slapped cheek" sign. An association with connective tissue disease (CTD) stigmata has recently been made. The clinical and dermatopathologic findings in 14 patients whose skin lesions were accompanied by serological evidence of B19 infection or documentation of B19 genome in lesional skin are presented. The authors encountered skin biopsy specimens from 14 patients who presented with skin eruptions accompanied by clinical signs or serology suggestive of antecedent B19 infection. Clinical findings were correlated to the light microscopic appearance of the lesions and the presence of B19 genome in lesional skin. The study group comprised 9 women, 3 men, and 2 boys. Eruptions characteristic of fifth disease, including the slapped cheek sign, reticulated truncal erythema, and acral petechiae, were present in 3 patients, 1 of whom later developed granuloma annulare. The other patients had atypical clinical presentations comprising an asymptomatic papular eruption (2), an eruption clinically resembling Sweet's syndrome (3), myopathic dermatomyositis (DM) (2), lupus erythematosus (LE)-like syndromes (2), and lower-extremity palpable purpura (2). Skin biopsy specimens in 12 cases showed interstitial histiocytic infiltrates with piecemeal fragmentation of collagen and a mononuclear cell-predominant vascular injury pattern. Other features included an interface dermatitis, eczematous alterations, and papillary dermal edema. Lesions with features of DM or LE also showed mesenchymal mucinosis, whereas a biopsied lesion of palpable purpura showed leukocytoclastic vasculitis (LCV). Immunofluorescent testing showed a positive lupus band test (LBT) with epidermal IgG and C5b-9 decoration in 1 patient with a systemic LE-like illness, whereas the DM patients had negative LBTs and vascular C5b-9

  12. Hereditary Spherocytosis Unmasked by Human Parvovirus B19 Induced Aplastic Crisis in a Family.

    PubMed

    Alavi, Samin; Arabi, Nahid; Yazdi, Mohammad Kaji; Arzanian, Mohammad Taghi; Zohrehbandian, Farahnaz

    2015-09-01

    Human parvovirus (HPV) B19 induced aplastic crisis in a family leading to the diagnosis of hereditary spherocytosis (HS) is a very rare condition being barely reported in the literature. We herein report a 4-year-old girl, her brother, and their mother who all presented with progressive pallor and jaundice after a febrile illness. The HPV B19 was diagnosed using polymerase chain reaction (PCR) and positive serology for specific anti-HPV B19 IgM. They were further diagnosed with having HS. The clinical importance of this report is that in the case of an abrupt onset of unexplained severe anemia and jaundice, one should consider underlying hemolytic anemias mostly hereditary spherocytosis complicated by HPV B19 aplastic crisis. Herein, we report the occurrence of this condition, simultaneously in three members of a family. The distinguished feature of this report is that all affected family members developed some degrees of transient pancytopenia, not only anemia, all simultaneously in the course of their disease. PMID:26379354

  13. 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

  14. Deciphering Host Genotype-Specific Impacts on the Metabolic Fingerprint of Listeria monocytogenes by FTIR Spectroscopy

    PubMed Central

    Grunert, Tom; Monahan, Avril; Lassnig, Caroline; Vogl, Claus; Müller, Mathias; Ehling-Schulz, Monika

    2014-01-01

    Bacterial pathogens are known for their wide range of strategies to specifically adapt to host environments and infection sites. An in-depth understanding of these adaptation mechanisms is crucial for the development of effective therapeutics and new prevention measures. In this study, we assessed the suitability of Fourier Transform Infrared (FTIR) spectroscopy for monitoring metabolic adaptations of the bacterial pathogen Listeria monocytogenes to specific host genotypes and for exploring the potential of FTIR spectroscopy to gain novel insights into the host-pathogen interaction. Three different mouse genotypes, showing different susceptibility to L. monocytogenes infections, were challenged with L. monocytogenes and re-isolated bacteria were subjected to FTIR spectroscopy. The bacteria from mice with different survival characteristics showed distinct IR spectral patterns, reflecting specific changes in the backbone conformation and the hydrogen-bonding pattern of the protein secondary structure in the bacterial cell. Coupling FTIR spectroscopy with chemometrics allowed us to link bacterial metabolic fingerprints with host infection susceptibility and to decipher longtime memory effects of the host on the bacteria. After prolonged cultivation of host-passaged bacteria under standard laboratory conditions, the host's imprint on bacterial metabolism vanished, which suggests a revertible metabolic adaptation of bacteria to host environment and loss of host environment triggered memory effects over time. In summary, our work demonstrates the potential and power of FTIR spectroscopy to be used as a fast, simple and highly discriminatory tool to investigate the mechanism of bacterial host adaptation on a macromolar and metabolic level. PMID:25541972

  15. Apolipoprotein E and Alzheimer disease: genotype-specific risks by age and sex.

    PubMed Central

    Bickeböller, H; Campion, D; Brice, A; Amouyel, P; Hannequin, D; Didierjean, O; Penet, C; Martin, C; Pérez-Tur, J; Michon, A; Dubois, B; Ledoze, F; Thomas-Anterion, C; Pasquier, F; Puel, M; Demonet, J F; Moreaud, O; Babron, M C; Meulien, D; Guez, D; Chartier-Harlin, M C; Frebourg, T; Agid, Y; Martinez, M; Clerget-Darpoux, F

    1997-01-01

    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 epsilon3 allele, an increased risk associated with the APOE epsilon4 allele (odds ratio [OR] [epsilon4] = 2.7 with 95% confidence interval [CI] = 2.0-3.6; P < .001) and a protective effect of the APOE epsilon2 allele (OR[epsilon2] = 0.5 with 95% CI = 0.3-0.98; P = .012) were retrieved. An effect of the epsilon4 allele dosage on susceptibility was confirmed (OR[epsilon4/epsilon4] vs. the epsilon3/epsilon3 genotype = 11.2 [95% CI = 4.0-31.6]; OR[epsilon3/epsilon4] vs. the epsilon3/epsilon3 genotype = 2.2 [95% CI = 1.5-3.5]). The frequency of the epsilon4 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 epsilon4 allele versus the epsilon3 allele, OR(epsilon4), were not equal in all age classes: OR(epsilon4) 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 epsilon3/epsilon4 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. PMID:9012418

  16. 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.

  17. Association of a functional Indoleamine 2,3-dioxygenase 2 genotype with specific immune responses

    PubMed Central

    Køllgaard, Tania; Klausen, Tobias Wirenfeldt; Idorn, Manja; Holmgaard, Rikke Bæk; Straten, Per thor; Andersen, Mads Hald

    2012-01-01

    Two frequent single-nucleotide-polymorphisms (SNPs) are present in the indoleamine 2,3-dioxygenase 2 (IDO2) gene that influence its enzymatic activity. Thus, one SNP (R248W) is associated with a reduction in IDO2 catalytic activity, whereas the other SNP (Y359stop) generates a premature stop codon abolishing activity completely. In the present study, we describe the presence of a specific cellular immune response in the periphery which correlated with the functional status of the IDO2 protein. Hence, the induction of IDO2-specific T cells in peripheral blood requires the presence of a functional IDO2 protein and, consequently, is restricted to individuals that are not homozygous for the stop codon. Furthermore, we detected stronger T-cell responses in donors with the homozygous Y wild type at position 359 when compared with the heterozygous genotype. Interestingly, we found a higher number of immune responses against IDO2 in patients homozygous for the 248W giving reduction in IDO2 activity compared with the 248R. Hence, spontaneous immune responses against IDO2 seem to be correlated with reduced enzymatic activity of IDO2. The patient IDO2 genotype may well influence the outcome of IDO2-based anti-cancer vaccination. PMID:22754762

  18. Postinfectious glomerulonephritis secondary to Erythrovirus B19 (Parvovirus B19): case report and review of the literature.

    PubMed

    Marco, Helena; Guermah, Imane; Matas, Lurdes; Hernández, Alba; Navarro, Maruja; Lopez, Dolores; Bonet, Josep

    2016-04-01

    A previously healthy 32-yearold woman developed arterial hypertension, proteinuria, and hematuria (nephritic syndrome) with normal renal function and was diagnosed with post-infectious glomerulonephritis secondary to parvovirus B19 infection. The renal biopsy showed endocapillary glomerulonephritis, with positive IgG, C3, and C1q immunoreactivity in the capillary walls and ultrastructural evidence of subendothelial deposits. The diagnosis of parvovirus B19 infection was confirmed by IgG/IgM serological positivity and parvovirus DNA demonstration in both peripheral blood and kidney tissue. Glomerular involvement improved spontaneously. To be noted are the atypical signs and symptoms of our patient who, unlike previously reported cases, failed to show fever, skin rash, or affected relatives. PMID:26833301

  19. Genotype-Specific Steroid Profiles Associated With Aldosterone-Producing Adenomas.

    PubMed

    Williams, Tracy Ann; Peitzsch, Mirko; Dietz, Anna S; Dekkers, Tanja; Bidlingmaier, Martin; Riester, Anna; Treitl, Marcus; Rhayem, Yara; Beuschlein, Felix; Lenders, Jacques W M; Deinum, Jaap; Eisenhofer, Graeme; Reincke, Martin

    2016-01-01

    Primary aldosteronism comprises 2 main subtypes: unilateral aldosterone-producing adenoma (APA) and bilateral adrenal hyperplasia. Somatic KCNJ5 mutations are found in APA at a prevalence of around 40% that drive and sustain aldosterone excess. Somatic APA mutations have been described in other genes (CACNA1D, ATP1A1, and ATP2B3) albeit at a lower frequency. Our objective was to identify genotype-specific steroid profiles in adrenal venous (AV) and peripheral venous (PV) plasma in patients with APAs. We measured the concentrations of 15 steroids in AV and PV plasma samples by liquid chromatography-tandem mass spectrometry from 79 patients with confirmed unilateral primary aldosteronism. AV sampling lateralization ratios of steroids normalized either to cortisol or to DHEA+androstenedione were also calculated. The hybrid steroid 18-oxocortisol exhibited 18- and 16-fold higher concentrations in lateralized AV and PV plasma, respectively, from APA with KCNJ5 mutations compared with all other APA combined together (P<0.001). Lateralization ratios for the KCNJ5 group were also generally higher. Strikingly, we demonstrate that a distinct steroid signature can differentiate APA genotype in AV and PV plasma. Notably, a 7-steroid fingerprint in PV plasma correctly classified 92% of the APA according to genotype. Prospective studies are necessary to translate these findings into clinical practice and determine if steroid fingerprinting could be of value to select patients with primary aldosteronism who are particularly suitable candidates for adrenal venous sampling because of a high probability of having an APA. PMID:26573708

  20. 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

  1. 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

  2. Immune response to B19 parvovirus and an antibody defect in persistent viral infection.

    PubMed Central

    Kurtzman, G J; Cohen, B J; Field, A M; Oseas, R; Blaese, R M; Young, N S

    1989-01-01

    B19 parvovirus has been shown to persist in some immunocompromised patients, and treatment with specific antibodies can lead to decreased quantities of circulating virus and hematologic improvement. A defective immune response to B19 parvovirus in these patients was shown by comparison of results using a capture RIA and immunoblotting. In normal individuals, examination of paired sera showed that the dominant humoral immune response during early convalescence was to the virus major capsid protein (58 kD) and during late convalescence to the minor capsid species (83 kD). In patients with persistent parvovirus infection, variable titers against intact particles were detected by RIA, but the sera from these patients had minimal or no IgG to capsid proteins determined by Western analysis. Competition experiments suggested that this discrepancy was not explicable on the basis of immune complex formation alone and that these patients may have a qualitative abnormality in antibody binding to virus. In neutralization experiments, in which erythroid colony formation in vitro was used as an assay of parvovirus activity, sera from patients with poor reactivity on immunoblotting were also inadequate in inhibiting viral infectivity. A cellular response to purified B19 parvovirus could not be demonstrated using proliferation assays and PBMC from individuals with serologic evidence of exposure to virus. These results suggest that production of neutralizing antibody to capsid protein plays a major role in limiting parvovirus infection in man. Images PMID:2551923

  3. 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

  4. Genotyping of Mycobacterium tuberculosis clinical isolates in two cities of Turkey: Description of a new family of genotypes that is phylogeographically specific for Asia Minor

    PubMed Central

    Zozio, Thierry; Allix, Caroline; Gunal, Selami; Saribas, Zeynep; Alp, Alpaslan; Durmaz, Riza; Fauville-Dufaux, Maryse; Rastogi, Nalin; Sola, Christophe

    2005-01-01

    Background Population-based bacterial genetics using repeated DNA loci is an efficient approach to study the biodiversity and phylogeographical structure of human pathogens, such as Mycobacterium tuberculosis, the agent of tuberculosis. Indeed large genetic diversity databases are available for this pathogen and are regularly updated. No population-based polymorphism data were yet available for M. tuberculosis in Turkey, at the crossroads of Eurasia. Results A total of 245 DNAs from Mycobacterium tuberculosis clinical isolates from tuberculosis patients residing in Turkey (Malatya n = 147 or Ankara n = 98) were genotyped by spoligotyping, a high-throughput genotyping method based on the polymorphism of the Direct Repeat locus. Thirty-three spoligotyping-defined clusters including 206 patients and 39 unique patterns were found. The ST41 cluster, as designated according to the international SpolDB3 database project, represented one fourth and when gathered to three genotypes, ST53, ST50 and ST284, one half of all the isolates. Out of 34 clinical isolates harboring ST41 which were further genotyped by IS6110 and by MIRU-VNTR typing, a typical 2-copy IS6110-RFLP pattern and a "215125113322" MIRU-VNTR pattern were observed among 21 clinical isolates. Further search in various databases confirms the likely Turkish-phylogeographical specificity of this clonal complex. Conclusion We described a new phylogeographically-specific clone of M. tuberculosis, designated LAM7-TUR. Further investigations to assess its frequency within all regions of Turkey and its phylogeographical origin and phylogenetic position within the global M. tuberculosis phylogenetic tree will shed new light on its endemicity in Asia Minor. PMID:16045794

  5. 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

  6. Pathologic characterization of genotypes XIV and XVII Newcastle disease viruses and efficacy of classical vaccination on specific pathogen-free birds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To characterize the clinico-pathological characteristics of recently-described genotypes of Newcastle disease virus (NDV), one representative strain of genotype XIV and two of genotype XVII, all isolated from West Africa, were used to infect four-week-old, specific pathogen free (SPF) chickens. The ...

  7. 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

  8. 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

  9. Acute Hepatitis as a Manifestation of Parvovirus B19 Infection ▿

    PubMed Central

    Hatakka, Aleisha; Klein, Julianne; He, Runtao; Piper, Jessica; Tam, Edward; Walkty, Andrew

    2011-01-01

    There are few reports in the literature of hepatitis as a manifestation of parvovirus B19 infection. We describe a case of parvovirus B19-associated acute hepatitis diagnosed based on a positive serologic test (IgM) and molecular detection of parvovirus B19 DNA in a liver biopsy specimen. Parvovirus B19 infection should be considered in the differential diagnosis of patients presenting with acute hepatitis. PMID:21734024

  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. PMID:27181213

  11. 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

  12. 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

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

    2016-04-01

    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

  13. 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

  14. 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

  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. 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

  17. 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

  18. Salinity stress in roots of contrasting barley genotypes reveals time-distinct and genotype-specific patterns for defined proteins.

    PubMed

    Witzel, Katja; Matros, Andrea; Strickert, Marc; Kaspar, Stephanie; Peukert, Manuela; Mühling, Karl H; Börner, Andreas; Mock, Hans-Peter

    2014-02-01

    Soil salinity is one of the most severe abiotic stress factors threatening agriculture worldwide. Hence, particular interest exists in unraveling mechanisms leading to salt tolerance and improved crop plant performance on saline soils. Barley is considered to be one of the most salinity-tolerant crops, but varying levels of tolerance are well characterized. A proteomic analysis of the roots of two contrasting cultivars (cv. Steptoe and cv. Morex) is presented. Young plants were exposed to a period of 1, 4, 7, or 10 d at 0, 100, or 150 mM NaCl. The root proteome was analyzed based on two-dimensional gel electrophoresis. A number of cultivar-specific and salinity stress-responsive proteins were identified. Mass spectrometry-based identification was successful for 74 proteins, and a hierarchical clustering analysis grouped these into five clusters based on similarity of expression profile. The rank product method was applied to statistically access the early and late responses, and this delivered a number of new candidate proteins underlying salinity tolerance in barley. Among these were some germin-like proteins, some pathogenesis-related proteins, and numerous as-yet uncharacterized proteins. Notably, proteins involved in detoxification pathways and terpenoid biosynthesis were detected as early responsive to salinity and may function as a means of modulating growth-regulating mechanisms and membrane stability via fine tuning of phytohormone and secondary metabolism in the root. PMID:24004485

  19. 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

  20. 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

  1. 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

  2. Different levels of food restriction reveal genotype-specific differences in learning a visual discrimination task.

    PubMed

    Makowiecki, Kalina; Hammond, Geoff; Rodger, Jennifer

    2012-01-01

    In behavioural experiments, motivation to learn can be achieved using food rewards as positive reinforcement in food-restricted animals. Previous studies reduce animal weights to 80-90% of free-feeding body weight as the criterion for food restriction. However, effects of different degrees of food restriction on task performance have not been assessed. We compared learning task performance in mice food-restricted to 80 or 90% body weight (BW). We used adult wildtype (WT; C57Bl/6j) and knockout (ephrin-A2⁻/⁻) mice, previously shown to have a reverse learning deficit. Mice were trained in a two-choice visual discrimination task with food reward as positive reinforcement. When mice reached criterion for one visual stimulus (80% correct in three consecutive 10 trial sets) they began the reverse learning phase, where the rewarded stimulus was switched to the previously incorrect stimulus. For the initial learning and reverse phase of the task, mice at 90%BW took almost twice as many trials to reach criterion as mice at 80%BW. Furthermore, WT 80 and 90%BW groups significantly differed in percentage correct responses and learning strategy in the reverse learning phase, whereas no differences between weight restriction groups were observed in ephrin-A2⁻/⁻ mice. Most importantly, genotype-specific differences in reverse learning strategy were only detected in the 80%BW groups. Our results indicate that increased food restriction not only results in better performance and a shorter training period, but may also be necessary for revealing behavioural differences between experimental groups. This has important ethical and animal welfare implications when deciding extent of diet restriction in behavioural studies. PMID:23144936

  3. Specific combinations of donor and recipient KIR-HLA genotypes predict for large differences in outcome after cord blood transplantation.

    PubMed

    Sekine, Takuya; Marin, David; Cao, Kai; Li, Li; Mehta, Pramod; Shaim, Hila; Sobieski, Catherine; Jones, Roy; Oran, Betul; Hosing, Chitra; Rondon, Gabriela; Alsuliman, Abdullah; Paust, Silke; Andersson, Borje; Popat, Uday; Kebriaei, Partow; Muftuoglu, Muharrem; Basar, Rafet; Kondo, Kayo; Nieto, Yago; Shah, Nina; Olson, Amanda; Alousi, Amin; Liu, Enli; Sarvaria, Anushruti; Parmar, Simrit; Armstrong-James, Darius; Imahashi, Nobuhiko; Molldrem, Jeffrey; Champlin, Richard; Shpall, Elizabeth J; Rezvani, Katayoun

    2016-07-14

    The ability of cord blood transplantation (CBT) to prevent relapse depends partly on donor natural killer (NK) cell alloreactivity. NK effector function depends on specific killer-cell immunoglobulin-like receptors (KIR) and HLA interactions. Thus, it is important to identify optimal combinations of KIR-HLA genotypes in donors and recipients that could improve CBT outcome. We studied clinical data, KIR and HLA genotypes, and NK-cell reconstitution in CBT patients (n = 110). Results were validated in an independent cohort (n = 94). HLA-KIR genotyping of recipient germline and transplanted cord blood (CB) grafts predicted for large differences in outcome. Patients homozygous for HLA-C2 group alleles had higher 1-year relapse rate and worse survival after CBT than did HLA-C1/C1 or HLA-C1/C2 (HLA-C1/x) patients: 67.8% vs 26.0% and 15.0% vs 52.9%, respectively. This inferior outcome was associated with delayed posttransplant recovery of NK cells expressing the HLA-C2-specific KIR2DL1/S1 receptors. HLA-C1/x patients receiving a CB graft with the combined HLA-C1-KIR2DL2/L3/S2 genotype had lower 1-year relapse rate (6.7% vs 40.1%) and superior survival (74.2% vs 41.3%) compared with recipients of grafts lacking KIR2DS2 or HLA-C1 HLA-C2/C2 patients had lower relapse rate (44.7% vs 93.4%) and better survival (30.1% vs 0%) if they received a graft with the combined HLA-C2-KIR2DL1/S1 genotype. Relapsed/refractory disease at CBT, recipient HLA-C2/C2 genotype, and donor HLA-KIR genotype were independent predictors of outcome. Thus, we propose the inclusion of KIR genotyping in graft selection criteria for CBT. HLA-C1/x patients should receive an HLA-C1-KIR2DL2/L3/S2 CB graft, while HLA-C2/C2 patients may benefit from an HLA-C2-KIR2DL1/S1 graft. PMID:27247137

  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. PMID:25073523

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

    PubMed Central

    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. 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. PMID:26210076

  7. Protective Effect of Human Leukocyte Antigen B27 in Hepatitis C Virus Infection Requires the Presence of a Genotype-Specific Immunodominant CD8+ T-Cell Epitope

    PubMed Central

    Kersting, Nadine; Fitzmaurice, Karen; Oniangue-Ndza, Cesar; Kemper, Michael N.; Humphreys, Isla; McKiernan, Susan; Kelleher, Dermot; Lohmann, Volker; Bowness, Paul; Huzly, Daniela; Rosen, Hugo R.; Kim, Arthur Y.; Lauer, Georg M.; Allen, Todd M.; Barnes, Eleanor; Roggendorf, Michael; Blum, Hubert E.; Thimme, Robert

    2015-01-01

    Human leukocyte antigen B27 (HLA-B27) is associated with protection in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection. This protective role is linked to single immunodominant HLA-B27-restricted CD8+ T-cell epitopes in both infections. In order to define the relative contribution of a specific HLA-B27-restricted epitope to the natural course of HCV infection, we compared the biological impact of the highly conserved HCV genotype 1 epitope, for which the protective role has been described, with the corresponding region in genotype 3 that differs in its sequence by three amino acid residues. The genotype 3a peptide was not recognized by CD8+ T cells specific for the genotype 1 peptide. Furthermore, patients with acute or chronic infection with HCV genotype 3a did not mount T-cell responses to this epitope region, and their autologous viral sequences showed no evidence of T-cell pressure. Finally, we found a significantly higher frequency of HLA-B27 positivity in patients with chronic HCV genotype 3a infection compared to genotype 1 infection, indicating that there is no protection by HLA-B27 in HCV genotype 3 infection. Conclusion Our data indicate that the protective effect of HLA-B27 is limited to HCV genotype 1 infection and does not expand to other genotypes such as genotype 3a. This can most likely be explained by intergenotype sequence diversity leading to the loss of the immunodominant HLA-B27 epitope in viral strains other than genotype 1. Our results underline the central role of a single HLA-B27-restricted epitope-specific CD8+ T-cell response in mediating protection in HCV genotype 1 infection. PMID:20034048

  8. 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

  9. 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

  10. Induction of apoptosis by human Nbk/Bik, a BH3-containing protein that interacts with E1B 19K.

    PubMed Central

    Han, J; Sabbatini, P; White, E

    1996-01-01

    The E1B 19-kilodalton protein (19K protein) is a potent apoptosis inhibitor and the adenovirus homolog of Bcl-2 (E. White, Genes Dev. 10:1-15, 1996). To obtain a better understanding of the biochemical mechanism by which the E1B 19K protein regulates apoptosis, proteins that interact with 19K have been identified; one of these is Bax (J. Han, P. Sabbatini, D. Perez, L. Rao, D. Mohda, and E. White, Genes Dev. 10:461-477, 1996), and another is Bak (S. N. Farrow, J. H. M. White, I. Martinou, T. Raven, K.-T. Pun, C. J. Grinham, J.-C. Martinou, and R. Brown, Nature (London) 374:731-733, 1995). Bax and Bak are Bcl-2 family members which contain Bcl-2 homology regions 1, 2, and 3 (BH1, BH2, and BH3), which interact with E1B 19K and Bcl-2 and promote apoptosis. Like Bax and Bak, Nbk was cloned from a yeast two-hybrid screen for proteins that interact with E1B 19K. Nbk contained BH3 but not BH1 or BH2. It also interacted with Bcl-2 but not with Bax. Both Bcl-2 and E1B 19K interacted with Nbk in vitro, and this interaction was highly specific. In vivo, the Nbk and E1B 19K proteins may colocalize with cytoplasmic and nuclear membranes. Nbk expression functionally antagonized 19K-mediated inhibition of apoptotic cell death and completely prevented transformation by E1A and E1B 19K. Nbk was sufficient for induction of apoptosis in the presence of mutant p53 and thus low levels of Bax, suggesting that Nbk functions independently of Bax to induce apoptosis. Nbk may therefore represent a novel death regulator which contains only a BH3 that interacts with and antagonizes apoptosis inhibitors such as the E1B 19K protein. PMID:8816500

  11. Acute encephalitis and encephalopathy associated with human parvovirus B19 infection in children.

    PubMed

    Watanabe, Toru; Kawashima, Hideshi

    2015-11-01

    Reports of neurologic manifestations of human parvovirus B19 (B19) infection have been on the rise. Acute encephalitis and encephalopathy is the most common, accounting for 38.8% of total B19-associated neurological manifestations. To date, 34 children with B19 encephalitis and encephalopathy have been reported, which includes 21 encephalitis and 13 encephalopathy cases. Ten (29%) were immunocompromised and 17 (39%) had underlying diseases. Fever at the onset of disease and rash presented in 44.1% and 20.6% of patients, respectively. Neurological manifestations include alteration of consciousness occurred in all patients, seizures in 15 (44.1%) patients, and focal neurologic signs in 12 (35.3%) patients. Anemia and pleocytosis in cerebrospinal fluid (CSF) occurred in 56.3% and 48.1% of patients, respectively. Serum Anti-B19 IgM (82.6%) and CSF B19 DNA (90%) were positive in the majority of cases. Some patients were treated with intravenous immunoglobulins and/or steroids, although an accurate evaluation of the efficacy of these treatment modalities cannot be determined. Nineteen (57.6%) patients recovered completely, 11 (33.3%) patients had some neurological sequelae and 3 (8.8%) patients died. Although the precise pathogenesis underlying the development of B19 encephalitis and encephalopathy is unclear, direct B19 infection or NS1protein of B19 toxicity in the brain, and immune-mediated brain injuries have been proposed. PMID:26566485

  12. Parvovirus B19-induced acute bilateral carpal tunnel syndrome in twin girls.

    PubMed

    Sakallı, Hale; Baskın, Esra; Dener, Şefik

    2015-01-01

    We describe 2 cases of 6-year-old twin girls presenting with acute carpal tunnel syndrome (CTS) associated with human parvovirus B19 (HPV-B19) infection, as evidenced by serological data and detection of HPV-B19 DNA in blood with use of polymerase chain reaction (PCR). To our knowledge, this is the first time that HPV-B19 infection has been suggested as the causal agent of simultaneous acute bilateral CTS in twins, thus presenting the possibility that similar immunologic responses can be observed in twins during viral infections. PMID:26422355

  13. Identification of G and P genotype-specific motifs in the predicted VP7 and VP4 amino acid sequences.

    PubMed

    Ma, Yongping

    2015-12-01

    Equine rotavirus (ERV) strain L338 (G13P[18]) has a unique G and P genotype. However, the evolutionary relationship of L338 with other ERVs is still unknown. Here whole genome analysis of the L338 ERV strain was independently performed. Its genotype constellations were determined as G13-P[18]-I6-R9-C9-M6-A6-N9-T12-E14-H11, confirming previous genotype assignments. The L338 strain only shared the P[18] and I6 genotypes with other ERVs. The nucleotide sequences of the other 9 RNA segments were different from those of cogent genes of all other group A rotavirus (RVA) strains including ERVs and formed unique phylogenetic lineages. The L338 evolutionary footprints were tentatively identified in both VP7 and VP4 amino acid sequences: two regions were found in VP7 and twelve in VP4. The conserved regions shared between L338 and other group A rotavirus strains (RVAs) indicated that L338 was more closely related genomically to animal and human RVAs other than ERVs, suggesting that L338 may not be an endogenous equine RV but have emerged as an interspecies reassortant with other RVA strains. Furthermore, genotype-specific motifs of all 27 G and 37 P types were identified in regions 7-1a (aa 91-100) of VP7 and regions 8-1 (aa146-151) and 8-3 (aa113-118 and 125-135) of VP4 (VP8*). PMID:26321159

  14. [Ballantyne syndrome caused by materno-fetal Parvovirus B19 infection: about two cases].

    PubMed

    Desvignes, F; Bourdel, N; Laurichesse-Delmas, H; Savary, D; Gallot, D

    2011-05-01

    Ballantyne's syndrome also known as Mirror syndrome is the association of fetal hydrops and maternal hydric retention. The maternal condition is often misdiagnosed as preeclampsia. We report two cases of Ballantyne syndrome associated with materno-fetal Parvovirus B19 infection. In the first case, the syndrome occurred at 26GW in a context of premature rupture of membranes. Parents and medical staff opted for termination of pregnancy because of the poor fetal prognosis. Maternal symptoms regressed after delivery. In the second case, the patient presented a Ballantyne's syndrome at 25GW. Intrauterine transfusions reversed symptomatology. Fetal hydrops of any etiology can be associated with this syndrome. Specific treatment of the fetus can avoid maternal complication allowing continuation of the pregnancy. PMID:21273007

  15. 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

  16. 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

  17. 29 CFR 2550.408b-19 - Statutory exemption for cross-trading of securities.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 29 Labor 9 2012-07-01 2012-07-01 false Statutory exemption for cross-trading of securities. 2550.408b-19 Section 2550.408b-19 Labor Regulations Relating to Labor (Continued) EMPLOYEE BENEFITS SECURITY ADMINISTRATION, DEPARTMENT OF LABOR FIDUCIARY RESPONSIBILITY UNDER THE EMPLOYEE RETIREMENT INCOME SECURITY ACT OF 1974 RULES AND REGULATIONS...

  18. Population Genomics Analysis of Legume Host Preference for Specific Rhizobial Genotypes in the Rhizobium leguminosarum bv. viciae Symbioses.

    PubMed

    Jorrin, Beatriz; Imperial, Juan

    2015-03-01

    Rhizobium leguminosarum bv. viciae establishes root nodule symbioses with several legume genera. Although most isolates are equally effective in establishing symbioses with all host genera, previous evidence suggests that hosts select specific rhizobial genotypes among those present in the soil. We have used population genomics to further investigate this observation. Pisum sativum, Lens culinaris, Vicia sativa, and V. faba plants were used to trap rhizobia from a well-characterized soil, and pooled genomic DNA from 100 isolates from each plant were sequenced. Sequence reads were aligned to the R. leguminosarum bv. viciae 3841 reference genome. High overall conservation of sequences was observed in all subpopulations, although several multigenic regions were absent from the soil population. A large fraction (16 to 22%) of sequence reads could not be recruited to the reference genome, suggesting that they represent sequences specific to that particular soil population. Although highly conserved, the 16S to 23S ribosomal RNA gene region presented single nucleotide polymorphisms (SNP) regarding the reference genome, but no striking differences could be found among plant-selected subpopulations. Plant-specific SNP patterns were, however, clearly observed within the nod gene cluster, supporting the existence of a plant preference for specific rhizobial genotypes. This was also shown after genome-wide analysis of SNP patterns. PMID:25514682

  19. Neurological aspects of human parvovirus B19 infection: a systematic review

    PubMed Central

    Barah, Faraj; Whiteside, Sigrid; Batista, Sonia; Morris, Julie

    2014-01-01

    Parvovirus B19 has been linked with various clinical syndromes including neurological manifestations. However, its role in the latter remains not completely understood. Although the last 10 years witnessed a surge of case reports on B19-associated neurological aspects, the literature data remains scattered and heterogeneous, and epidemiological information on the incidence of B19-associated neurological aspects cannot be accurately extrapolated. The aim of this review is to identify the characteristics of cases of B19-associated neurological manifestations. A computerized systematic review of existing literature concerning cases of B19-related neurological aspects revealed 89 articles describing 129 patients; 79 (61.2%) were associated with CNS manifestations, 41 (31.8%) were associated with peripheral nervous system manifestations, and 9 (7.0%) were linked with myalgic encephalomyelitis. The majority of the cases (50/129) had encephalitis. Clinical characteristic features of these cases were analyzed, and possible pathological mechanisms were also described. In conclusion, B19 should be included in differential diagnosis of encephalitic syndromes of unknown etiology in all age groups. Diagnosis should rely on investigation of anti-B19 IgM antibodies and detection of B19 DNA in serum or CSF. Treatment of severe cases might benefit from a combined regime of intravenous immunoglobulins and steroids. To confirm these outcomes, goal-targeted studies are recommended to exactly identify epidemiological scenarios and explore potential pathogenic mechanisms of these complications. Performing retrospective and prospective and multicenter studies concerning B19 and neurological aspects in general, and B19 and encephalitic syndromes in particular, are required. © 2014 The Authors. Reviews in Medical Virology published by John Wiley & Sons, Ltd. PMID:24459081

  20. Host-specificity of Staphylococcus aureus causing intramammary infections in dairy animals assessed by genotyping and virulence genes.

    PubMed

    Bar-Gal, G Kahila; Blum, S E; Hadas, L; Ehricht, R; Monecke, S; Leitner, G

    2015-03-23

    Staphylococcus aureus is one of the most relevant pathogens causing clinical and subclinical, chronic mastitis in dairy animals. Routinely, mastitis pathogens are isolated and classified to genus or species level, and regarded as single entities. However, S. aureus includes a broad range of genotypes with distinct pathogenic and epidemiologic characteristics. The objective of the present study was to assess the host-specificity of S. aureus causing mastitis in dairy animals, based on phylogenetic and genotypic characterization as well as the presence of virulence and antimicrobial resistance genes in the pathogen genome. S. aureus isolates from mastitis in cows, sheep and goats in Israel, and from cows in Germany, the USA and Italy, were compared by the following methods: a. Bayesian phylogenetic comparison of sequences of genes nuc, coa, lukF and clfA, b. genotyping by spa and agr typing, and assignment to MLST Clonal Complexes (MLST CC), and c. the presence of a broad array of virulence and antimicrobial resistance genes. Overall, phylogenetic, virulence and genotyping approaches agreed with each other. Cow isolates could be differentiated from sheep and goat isolates with all three methods, with different resolution. In two phylogenetic clusters, segregation was found also between cow isolates from Israel and abroad. Sheep and goats' isolates showed less variability than isolates from cows in all methods used. In conclusion, different S. aureus lineages are associated to cows in contrast to goats and sheep, suggesting co-evolution between pathogen and host species. Modern diagnostics approaches should aim to explore molecular data for a better understanding and cost-effective management of mastitis. PMID:25631254

  1. 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

  2. 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 Central

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

    1986-01-01

    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. Images PMID:3021988

  3. Patch-Clamp Study of Hepatitis C p7 Channels Reveals Genotype-Specific Sensitivity to Inhibitors.

    PubMed

    Breitinger, Ulrike; Farag, Noha S; Ali, Nourhan K M; Breitinger, Hans-Georg A

    2016-06-01

    Hepatitis C is a major worldwide disease and health hazard, affecting ∼3% of the world population. The p7 protein of hepatitis C virus (HCV) is an intracellular ion channel and pH regulator that is involved in the viral replication cycle. It is targeted by various classical ion channel blockers. Here, we generated p7 constructs corresponding to HCV genotypes 1a, 2a, 3a, and 4a for recombinant expression in HEK293 cells, and studied p7 channels using patch-clamp recording techniques. The pH50 values for recombinant p7 channels were between 6.0 and 6.5, as expected for proton-activated channels, and current-voltage dependence did not show any differences between genotypes. Inhibition of p7-mediated currents by amantadine, however, exhibited significant, genotype-specific variation. The IC50 values of p7-1a and p7-4a were 0.7 ± 0.1 nM and 3.2 ± 1.2 nM, whereas p7-2a and p7-3a had 50- to 1000-fold lower sensitivity, with IC50 values of 2402 ± 334 nM and 344 ± 64 nM, respectively. The IC50 values for rimantadine were low across all genotypes, ranging from 0.7 ± 0.1 nM, 1.6 ± 0.6 nM, and 3.0 ± 0.8 nM for p7-1a, p7-3a, and p7-4a, respectively, to 24 ± 4 nM for p7-2a. Results from patch-clamp recordings agreed well with cellular assays of p7 activity, namely, measurements of intracellular pH and hemadsorption assays, which confirmed the much reduced amantadine sensitivity of genotypes 2a and 3a. Thus, our results establish patch-clamp studies of recombinant viroporins as a valid analytical tool that can provide quantitative information about viroporin channel properties, complementing established techniques. PMID:27276260

  4. Use of allele-specific FAIRE to determine functional regulatory polymorphism using large-scale genotyping arrays.

    PubMed

    Smith, Andrew J P; Howard, Philip; Shah, Sonia; Eriksson, Per; Stender, Stefan; Giambartolomei, Claudia; Folkersen, Lasse; Tybjærg-Hansen, Anne; Kumari, Meena; Palmen, Jutta; Hingorani, Aroon D; Talmud, Philippa J; Humphries, Steve E

    2012-01-01

    Following the widespread use of genome-wide association studies (GWAS), focus is turning towards identification of causal variants rather than simply genetic markers of diseases and traits. As a step towards a high-throughput method to identify genome-wide, non-coding, functional regulatory variants, we describe the technique of allele-specific FAIRE, utilising large-scale genotyping technology (FAIRE-gen) to determine allelic effects on chromatin accessibility and regulatory potential. FAIRE-gen was explored using lymphoblastoid cells and the 50,000 SNP Illumina CVD BeadChip. The technique identified an allele-specific regulatory polymorphism within NR1H3 (coding for LXR-α), rs7120118, coinciding with a previously GWAS-identified SNP for HDL-C levels. This finding was confirmed using FAIRE-gen with the 200,000 SNP Illumina Metabochip and verified with the established method of TaqMan allelic discrimination. Examination of this SNP in two prospective Caucasian cohorts comprising 15,000 individuals confirmed the association with HDL-C levels (combined beta = 0.016; p = 0.0006), and analysis of gene expression identified an allelic association with LXR-α expression in heart tissue. Using increasingly comprehensive genotyping chips and distinct tissues for examination, FAIRE-gen has the potential to aid the identification of many causal SNPs associated with disease from GWAS. PMID:22916038

  5. 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

  6. Genome-wide association analyses of child genotype effects and parent-of-origin effects in specific language impairment

    PubMed Central

    Nudel, R; Simpson, N H; Baird, G; O’Hare, A; Conti-Ramsden, G; Bolton, P F; Hennessy, E R; Ring, S M; Davey Smith, G; Francks, C; Paracchini, S; Monaco, A P; Fisher, S E; Newbury, D F

    2014-01-01

    Specific language impairment (SLI) is a neurodevelopmental disorder that affects linguistic abilities when development is otherwise normal. We report the results of a genome-wide association study of SLI which included parent-of-origin effects and child genotype effects and used 278 families of language-impaired children. The child genotype effects analysis did not identify significant associations. We found genome-wide significant paternal parent-of-origin effects on chromosome 14q12 (P = 3.74 × 10−8) and suggestive maternal parent-of-origin effects on chromosome 5p13 (P = 1.16 × 10−7). A subsequent targeted association of six single-nucleotide-polymorphisms (SNPs) on chromosome 5 in 313 language-impaired individuals and their mothers from the ALSPAC cohort replicated the maternal effects, albeit in the opposite direction (P = 0.001); as fathers’ genotypes were not available in the ALSPAC study, the replication analysis did not include paternal parent-of-origin effects. The paternally-associated SNP on chromosome 14 yields a non-synonymous coding change within the NOP9 gene. This gene encodes an RNA-binding protein that has been reported to be significantly dysregulated in individuals with schizophrenia. The region of maternal association on chromosome 5 falls between the PTGER4 and DAB2 genes, in a region previously implicated in autism and ADHD. The top SNP in this association locus is a potential expression QTL of ARHGEF19 (also called WGEF) on chromosome 1. Members of this protein family have been implicated in intellectual disability. In summary, this study implicates parent-of-origin effects in language impairment, and adds an interesting new dimension to the emerging picture of shared genetic etiology across various neurodevelopmental disorders. PMID:24571439

  7. 29 CFR 2550.408b-19 - Statutory exemption for cross-trading of securities.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... exemption under section 408(b)(19) of the Act requires satisfaction of several objective conditions in... customer or pooled fund or account. (2) The term “compliance officer” means an individual designated by...

  8. [The course of parvovirus B19 infection during pregnancy after kidney transplantation].

    PubMed

    Mavrina, E S; Garmaeva, T Ts; Troitskaia, V V; Makhinia, S A; Glinshchikova, O A; Biriukova, L S; Mikhaĭlova, E A; Parovichnikova, E N; Savchenko, V G

    2013-01-01

    Having a tropism for erythroid progenitor cells, parvovirus B19 may cause partial red cell aplasia and thrombocytopenia. Early diagnosis of parvovirus B19 infection in immunocompromised patients is needed for timely antiviral therapy. A high-risk group for parvovirus B19 infection includes patients with blood diseases who receive multiple transfusions of blood components; those who have undergone donor organ transplantation and are long taking immunosuppressive drugs; and pregnant women. These patients require careful virological monitoring for major blood-borne viral infections. This paper describes a clinical case of parvovirus B19 infection in a pregnant woman who has undergone kidney transplantation and is continuously taking immunosuppressive medications. Identification of the cause of severe anemia and timely adequate therapy could lead to the recovery of effective erythropoiesis in the patient. PMID:24432604

  9. Focal epilepsy as a long term sequela of Parvovirus B19 encephalitis.

    PubMed

    Palermo, Concetta Ilenia; Costanzo, Carmela Maria; Franchina, Concetta; Castiglione, Giacomo; Giuliano, Loretta; Russo, Raffaela; Conti, Alessandro; Sofia, Vito; Scalia, Guido

    2016-07-01

    Human Parvovirus B19 (PVB19), the etiological agent of the fifth disease, is associated with a large spectrum of pathologies, among which is encephalitis. Since it has been detected from the central nervous system in children or in immunocompromised patients, its causative role in serious neurological manifestations is still unclear. Here we report the case of an 18-year-old healthy boy who developed encephalitis complicated by prolonged status epilepticus. The detection of PVB19 DNA in his serum and, subsequently, in his cerebrospinal fluid supports the hypothesis that this virus could potentially play a role in the pathogenesis of neurological complications. In addition, the detection of viral DNA and the presence of specific IgM and IgG antibodies in serum, together with clinical findings such as skin rash, support the presence of a disseminated viral infection. In the presence of neurological disorders, especially when there are no specific signs, but seizures and rash are present, it is important to search for PVB19 both in immunocompromised and immunocompetent patients. Moreover, the introduction of the PVB19 DNA test into diagnostic protocols of neuropathies, especially those undiagnosed, could clarify the etiological agent that otherwise could remain unrecognized. PMID:27130981

  10. 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

  11. Splenic infarcts as a rare manifestation of parvovirus B19 infection

    PubMed Central

    Kranidiotis, Georgios; Efstratiadis, Efrosini; Kapsalakis, Georgios; Loizos, Georgios; Bilis, Apostolos; Melidonis, Andreas

    2016-01-01

    Introduction Human parvovirus B19 is a DNA virus most known for causing erythema infectiosum in children, and polyarthropathy or transient aplastic crisis in adults. However, various unusual clinical manifestations have also been reported in association with it. We describe a young patient who presented with splenic infarcts as a rare complication of B19 infection. Case report A 33-year old previously healthy man was admitted to our hospital because of a 5-day history of fever and headache. Imaging studies revaled two splenic infarcts. Endocarditis was ruled out, whereas serologic testing for B19 was indicative of acute infection. Discussion To our knowledge, three cases of thromboembolism in the setting of B19 infection have been reported up to now, including one occurence of splenic infarction. These events were attributed to the development of a transient antiphospholipid antibody syndrome. In contrast, our patient did not have elevated titers of antiphospholipid antibodies. Conclusions Splenic infarcts can be an atypical presentation of B19 infection. Parvovirus B19 may induce thromboembolic events, even in the absence of antiphospholipid antibodies. PMID:27213135

  12. 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.

  13. 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

  14. The role of parvovirus B19 and the immune response in the pathogenesis of acute leukemia.

    PubMed

    Kerr, Jonathan R; Mattey, Derek L

    2015-05-01

    In this article, we review the evidence suggesting a possible role for B19 virus in the pathogenesis of a subset of cases of acute leukemia. Human parvovirus B19 infection may complicate the clinical course of patients with acute leukemia and may also precede the development of acute leukemia by up to 180 days. Parvovirus B19 targets erythroblasts in the bone marrow and may cause aplastic crisis in patients with shortened-red cell survival. Aplastic crisis represents a prodrome of acute lymphoblastic leukemia in 2% patients. There is a significant overlap between those HLA classes I and II alleles that are associated with a vigorous immune response and development of symptoms during B19 infection and those HLA alleles that predispose to development of acute leukemia. Acute symptomatic B19 infection is associated with low circulating IL-10 consistent with a vigorous immune response; deficient IL-10 production at birth was recently found to be associated with subsequent development of acute leukemia. Anti-B19 IgG has been associated with a particular profile of methylation of human cancer genes in patients with acute leukemia, suggesting an additional hit and run mechanism. The proposed role for parvovirus B19 in the pathogenesis of acute leukemia fits well with the delayed infection hypothesis and with the two-step mutation model, which describes carriage of the first mutation prior to birth, followed by suppression of hematopoiesis, which allows rapid proliferation of cells harboring the first mutation, acquisition of a second activating mutation, and expansion of cells carrying both mutations, resulting in acute leukemia. PMID:25855476

  15. Unscrambling the role of human parvovirus B19 signaling in systemic autoimmunity.

    PubMed

    Tsay, Gregory J; Zouali, Moncef

    2006-11-30

    Despite enormous progress in understanding how the immune system works, the pathogenesis of autoimmune diseases still remains unclear. Growing evidence indicates that infectious agents can be potent initial triggers, subverting and exploiting host cell signaling pathways. This role is exemplified by the association of parvovirus B19 (B19) with human autoimmune disease. Infection with this common virus exhibits striking similarities with systemic autoimmune diseases, and can be associated with elevated serum autoantibody titers. The B19 virus produces proline-rich, 11-kDa proteins that have been implicated in modulation of host signaling cascades involved in virulence and pathogenesis. Additionally, B19 produces a non-structural protein (NS1) that functions as a transcription regulator by directly binding the p6 promoter and the Sp1/Sp3 transcription factors. The protein is also involved in DNA replication, cell cycle arrest and initiation of apoptotic damage, particularly in erythroid cells. When transfected to non-permissive cells, NS1 recruits the mitochondria cell death pathway. It is even more remarkable that NS1 functions as a trans-acting transcription activator for the IL6 promoter, up-regulating IL6 expression in host cells. Hence, B19 infection may play a pivotal role in triggering inflammatory disorders. By promoting apoptotic damage and trans-activating pro-inflammatory cytokine promoters, B19 may break the delicate balance between cell survival and apoptosis, and may contribute to immune deregulation. Understanding the mechanisms used by B19 to alter the cell signaling machinery may provide further insight into the mechanism by which autoimmune diseases develop. PMID:16764828

  16. Strain- and genotype-specific differences in virulence of Paenibacillus larvae subsp. larvae, a bacterial pathogen causing American foulbrood disease in honeybees.

    PubMed

    Genersch, Elke; Ashiralieva, Ainura; Fries, Ingemar

    2005-11-01

    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

  17. 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

  18. Quantitative genotyping of mouse brain-specific PEX13 gene disruption by real-time PCR.

    PubMed

    Müller, C Catharina; Nourse, Jamie P; Nguyen, Tam H; Crane, Denis I

    2009-06-30

    The Cre/loxP-system has become an invaluable tool for the generation of tissue-specific gene disruption in mice. However, because Cre recombinase excision of individual genes can be variable, an accurate and sensitive method is necessary to determine the ultimate level of gene disruption. The analysis of gene disruption is particularly difficult for tissue that has been fixed for (immuno)histochemical analysis with paraformaldehyde. Here, we describe a simple, rapid and cost effective method for measurement of gene disruption using quantitative real-time PCR, through application to the analysis of PEX13 gene disruption in a brain-specific PEX13 mouse mutant. We show that this general protocol is suitable for both normal and paraformaldehyde-fixed tissue. PMID:19422853

  19. 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

  20. 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

  1. High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities.

    PubMed

    Hart, Traver; Chandrashekhar, Megha; Aregger, Michael; Steinhart, Zachary; Brown, Kevin R; MacLeod, Graham; Mis, Monika; Zimmermann, Michal; Fradet-Turcotte, Amelie; Sun, Song; Mero, Patricia; Dirks, Peter; Sidhu, Sachdev; Roth, Frederick P; Rissland, Olivia S; Durocher, Daniel; Angers, Stephane; Moffat, Jason

    2015-12-01

    The ability to perturb genes in human cells is crucial for elucidating gene function and holds great potential for finding therapeutic targets for diseases such as cancer. To extend the catalog of human core and context-dependent fitness genes, we have developed a high-complexity second-generation genome-scale CRISPR-Cas9 gRNA library and applied it to fitness screens in five human cell lines. Using an improved Bayesian analytical approach, we consistently discover 5-fold more fitness genes than were previously observed. We present a list of 1,580 human core fitness genes and describe their general properties. Moreover, we demonstrate that context-dependent fitness genes accurately recapitulate pathway-specific genetic vulnerabilities induced by known oncogenes and reveal cell-type-specific dependencies for specific receptor tyrosine kinases, even in oncogenic KRAS backgrounds. Thus, rigorous identification of human cell line fitness genes using a high-complexity CRISPR-Cas9 library affords a high-resolution view of the genetic vulnerabilities of a cell. PMID:26627737

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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 Central

    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

  8. 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

  9. Effects of human parvovirus B19 on expression of defensins and Toll-like receptors.

    PubMed

    Hsu, Gwo-Jong; Tzang, Bor-Show; Tsai, Chun-Chou; Chiu, Chun-Ching; Huang, Chih-Yang; Hsu, Tsai-Ching

    2011-10-31

    Both cell-mediated and humoral immunity have been widely investigated for the roles in pathogenesis of human parvovirus B19 (B19) infection. However, little is known about the effects of B19 infection on innate immunity. In the current study, expression of alpha-human neutrophil peptides (HNP) 1-3, alpha-human defensin (HD) 5, HD6, beta-human defensin (hBD)-1, hBD-3, toll-like receptor (TLR) 4, TLR5, TLR7 and TLR9 in B19-nonstructural protein (NS)-1 or B19-viral protein (VP)-2 transfected COS-7 cells was investigated by reverse transcription (RT)-PCR or by western blots. Significantly increased HNP1-3, HD5, HD6, hBD1 and hBD3 mRNA levels were detected at both 24 h and 20 days post-transfection in COS-7 cells transfected with pEGFP-NS1. In pEGFP-VP2-transfected COS-7 cells, significantly increased HNP1-3, HD5, HD6, hBD-1 and hBD-3 mRNA expression levels were observed on day 20, albeit only hBD3 mRNA increased significantly at 24 h post-transfection. Additionally, TLR4, TLR5 and TLR7 proteins decreased significantly in COS-7 cells transfected with pEGFP-NS1 or pEGFP-VP2 at 48 h but significantly increased on day 20. Notably, only TLR9 protein increased significantly in the cells transfected with pEGFP-NS1 on day 20. No significant variation of TLRs was observed in cells transfected with pEGFP-NS1K334E, a single substitution mutantation of B19-NS1 protein without original cytotoxicity, at both 48 h and on day 20. These novel findings revealed the different effects of B19-NS1 and VP2 on the stimulation of defensins and TLRs and could provide a clue in understanding the roles of B19-NS1 and VP2 on innate immunity. PMID:22135916

  10. Rheumatologic manifestations of human parvovirus B19 infection in adults. Initial two-year clinical experience.

    PubMed

    Naides, S J; Scharosch, L L; Foto, F; Howard, E J

    1990-09-01

    During 1987 and 1988, we identified 9 adults at the Medical and Rheumatology Services of the University of Iowa Hospitals and Clinics who had a clinical diagnosis of fifth disease; 8 of the 9 had symptoms of joint involvement. Another 12 adults with serologic positivity for anti-parvovirus B19 IgM antibody presented with polyarthralgia/polyarthritis. Patients were usually found to be seronegative for rheumatoid factor, and none developed nodules or erosive disease. Many patients with chronic disease met criteria for a diagnosis of rheumatoid arthritis. A diagnosis of parvovirus B19 infection should be considered during the initial visit of patients with polyarthralgia/polyarthritis. PMID:2169746

  11. 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)...

  12. 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

  13. 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. PMID:23840852

  14. 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

  15. 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. PMID:24191900

  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. [Comparative study of indirect immunofluorescence (IIF) and ELISA techniques in the detection of parvovirus B19].

    PubMed

    González, M; Hassanhi, M; Rivera, S; Bracho, M P

    2000-03-01

    To determine the seroprevalence of IgG and IgM antibodies against Parvovirus B19 (P. B19), we studied the sera of 53 patients with different hematologic disorders and the sera of 15 controls using indirect immunofluorescence (IFI) and the ELISA method. The prevalence of IgG in the control group was 46.6%, in patients with aplastic crisis was 83.3% (IFI) and 66.7% (ELISA) and, in patients without crisis was 68.9% (IFI) and 72.4% (ELISA). IgM was negative except for patients with crisis: 8.3% (IFI) and 29.1% (ELISA). The higher seroprevalence (IgG) found in patients in comparison with controls might be due to a greater exposure of of patients to the virus. The agreement for both techniques was 81%(IgG) and 93% (IgM) however ELISA technique was more sensitive for detecting IgM of P. B19. In spite of serologic evidence and evaluating a simple serum sample per patient, we could establish an association between aplastic crisis and viral infection for IgM ELISA but not for IgG between hematologic disorders and infection for the P. B19. PMID:10758696

  18. 26 CFR 31.3121(b)(19)-1 - Services of certain nonresident aliens.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 26 Internal Revenue 15 2012-04-01 2012-04-01 false Services of certain nonresident aliens. 31.3121... 1954) General Provisions § 31.3121(b)(19)-1 Services of certain nonresident aliens. (a) (1) Services performed after 1961 by a nonresident alien individual who is temporarily present in the United States as...

  19. 26 CFR 31.3121(b)(19)-1 - Services of certain nonresident aliens.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 26 Internal Revenue 15 2014-04-01 2014-04-01 false Services of certain nonresident aliens. 31.3121... 1954) General Provisions § 31.3121(b)(19)-1 Services of certain nonresident aliens. (a) (1) Services performed after 1961 by a nonresident alien individual who is temporarily present in the United States as...

  20. Mononeuropathy multiplex associated with acute parvovirus B19 infection: characteristics, treatment and outcome.

    PubMed

    Lenglet, Timothée; Haroche, Julien; Schnuriger, Aurélie; Maisonobe, Thierry; Viala, Karine; Michel, Yanne; Chelbi, Farhat; Grabli, David; Seror, Paul; Garbarg-Chenon, Antoine; Amoura, Zahir; Bouche, Pierre

    2011-07-01

    To describe the characteristics of peripheral neuropathy related to acute parvovirus B19 (B19V) infection. We reviewed clinical, electrophysiological and histological data of three patients with peripheral neuropathy and positive B19V detection (IgG, IgM and PCR) compatible with acute infection. The neuropathy fulfilled criteria for mononeuropathy multiplex (MM). It could be preceded by or concurrent with a limited purpuric eruption, but systemic manifestations were absent. The first neurological symptoms were always sensory and localized in a hand. Neuropathy was initially limited to a restricted sensory part of a nerve trunk territory. The course was subacute with successive and asymmetric injury of the limb and cranial nerves. Electromyographic study confirmed the diagnosis of MM with multifocal asymmetric sensory and motor axonal loss in two patients, whereas the neuropathy was purely sensory and limited to two nerves in the other patient. Nerve biopsies showed no evidence of necrotizing vasculitis but, in one patient, revealed a lymphocytic perivascular infiltrate evocative of hypersensitivity vasculitis secondary to an infectious agent. Intravenous immunoglobulin (IVIg) was systematically administered. Long-term outcome was good but with incomplete sensory recovery and, for one patient, persistence of a functional disability. B19 V infection should be considered in the etiological assessment of MM, especially in the event of a progressive sensory disorder in the hands and a concomitant history of rash. IVIg may be an effective treatment for this inflammatory disorder. PMID:21287183

  1. Delay-specific stimuli and genotype interact to determine temporal discounting in a rapid-acquisition procedure.

    PubMed

    Pope, Derek A; Newland, M Christopher; Hutsell, Blake A

    2015-05-01

    The importance of delay discounting to many socially important behavior problems has stimulated investigations of biological and environmental mechanisms responsible for variations in the form of the discount function. The extant experimental research, however, has yielded disparate results, raising important questions regarding Gene X Environment interactions. The present study determined the influence of stimuli that uniquely signal delays to reinforcement on delay discounting in two inbred mouse strains using a rapid-acquisition procedure. BALB/c and C57BL/6 mice responded under a six-component, concurrent-chained schedule in which the terminal-link delays preceding the larger-reinforcer were presented randomly across components of an individual session. Across conditions, components were presented either with or without delay-specific auditory stimuli, i.e., as multiple or mixed schedules. A generalized matching-based model was used to incorporate the impact of current and previous component reinforcer-delay ratios on current component response allocation. Sensitivity to reinforcer magnitude and delay were higher for BALB/c mice, but within-component preference reached final levels faster for C57Bl/6 mice. For BALB/c mice, acquisition of preference across blocks of a component was faster under the multiple than the mixed schedule, but final levels of sensitivity to reinforcement were unaffected by schedule. The speed of acquisition of preference was not different across schedules for C57Bl/6 mice, but sensitivity to reinforcement was higher under the multiple than the mixed schedule. Overall, differences in the acquisition and final form of the discount function were determined by a Gene X Environment interaction, but the presence of delay-specific stimuli attenuated genotype-dependent differences in magnitude and delay sensitivity. PMID:25869302

  2. 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.

  3. Specific and straightforward molecular investigation of β-thalassemia mutations in the Malaysian Malays and Chinese using direct TaqMan genotyping assays.

    PubMed

    Kho, S L; Chua, K H; George, E; Tan, J A M A

    2013-01-01

    Beta-thalassemia is a life-threatening inherited blood disorder. Rapid characterization of β-globin gene mutations is necessary because of the high frequency of Malaysian β-thalassemia carriers. A combination real-time polymerase chain reaction genotyping assay using TaqMan probes was developed to confirm β-globin gene mutations. In this study, primers and probes were designed to specifically identify 8 common β-thalassemia mutations in the Malaysian Malay and Chinese ethnic groups using the Primer Express software. "Blind tests" using DNA samples from healthy individuals and β-thalassemia patients with different genotypes were performed to determine the specificity and sensitivity of this newly designed assay. Our results showed 100% sensitivity and specificity for this novel assay. In conclusion, the TaqMan genotyping assay is a straightforward assay that allows detection of β-globin gene mutations in less than 40 min. The simplicity and reproducibility of the TaqMan genotyping assay permit its use in laboratories as a rapid and cost-effective diagnostic tool for confirmation of common β-thalassemia mutations in Malaysia. PMID:23479149

  4. 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

  5. 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. PMID:26632342

  6. 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. PMID:23555760

  7. 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. PMID:25462010

  8. Rare variant genotype imputation with thousands of study-specific whole-genome sequences: implications for cost-effective study designs

    PubMed Central

    Pistis, Giorgio; Porcu, Eleonora; Vrieze, Scott I; Sidore, Carlo; Steri, Maristella; Danjou, Fabrice; Busonero, Fabio; Mulas, Antonella; Zoledziewska, Magdalena; Maschio, Andrea; Brennan, Christine; Lai, Sandra; Miller, Michael B; Marcelli, Marco; Urru, Maria Francesca; Pitzalis, Maristella; Lyons, Robert H; Kang, Hyun M; Jones, Chris M; Angius, Andrea; Iacono, William G; Schlessinger, David; McGue, Matt; Cucca, Francesco; Abecasis, Gonçalo R; Sanna, Serena

    2015-01-01

    The utility of genotype imputation in genome-wide association studies is increasing as progressively larger reference panels are improved and expanded through whole-genome sequencing. Developing general guidelines for optimally cost-effective imputation, however, requires evaluation of performance issues that include the relative utility of study-specific compared with general/multipopulation reference panels; genotyping with various array scaffolds; effects of different ethnic backgrounds; and assessment of ranges of allele frequencies. Here we compared the effectiveness of study-specific reference panels to the commonly used 1000 Genomes Project (1000G) reference panels in the isolated Sardinian population and in cohorts of European ancestry including samples from Minnesota (USA). We also examined different combinations of genome-wide and custom arrays for baseline genotypes. In Sardinians, the study-specific reference panel provided better coverage and genotype imputation accuracy than the 1000G panels and other large European panels. In fact, even gene-centered custom arrays (interrogating ~200 000 variants) provided highly informative content across the entire genome. Gain in accuracy was also observed for Minnesotans using the study-specific reference panel, although the increase was smaller than in Sardinians, especially for rare variants. Notably, a combined panel including both study-specific and 1000G reference panels improved imputation accuracy only in the Minnesota sample, and only at rare sites. Finally, we found that when imputation is performed with a study-specific reference panel, cutoffs different from the standard thresholds of MACH-Rsq and IMPUTE-INFO metrics should be used to efficiently filter badly imputed rare variants. This study thus provides general guidelines for researchers planning large-scale genetic studies. PMID:25293720

  9. Rare variant genotype imputation with thousands of study-specific whole-genome sequences: implications for cost-effective study designs.

    PubMed

    Pistis, Giorgio; Porcu, Eleonora; Vrieze, Scott I; Sidore, Carlo; Steri, Maristella; Danjou, Fabrice; Busonero, Fabio; Mulas, Antonella; Zoledziewska, Magdalena; Maschio, Andrea; Brennan, Christine; Lai, Sandra; Miller, Michael B; Marcelli, Marco; Urru, Maria Francesca; Pitzalis, Maristella; Lyons, Robert H; Kang, Hyun M; Jones, Chris M; Angius, Andrea; Iacono, William G; Schlessinger, David; McGue, Matt; Cucca, Francesco; Abecasis, Gonçalo R; Sanna, Serena

    2015-07-01

    The utility of genotype imputation in genome-wide association studies is increasing as progressively larger reference panels are improved and expanded through whole-genome sequencing. Developing general guidelines for optimally cost-effective imputation, however, requires evaluation of performance issues that include the relative utility of study-specific compared with general/multipopulation reference panels; genotyping with various array scaffolds; effects of different ethnic backgrounds; and assessment of ranges of allele frequencies. Here we compared the effectiveness of study-specific reference panels to the commonly used 1000 Genomes Project (1000G) reference panels in the isolated Sardinian population and in cohorts of European ancestry including samples from Minnesota (USA). We also examined different combinations of genome-wide and custom arrays for baseline genotypes. In Sardinians, the study-specific reference panel provided better coverage and genotype imputation accuracy than the 1000G panels and other large European panels. In fact, even gene-centered custom arrays (interrogating ~200 000 variants) provided highly informative content across the entire genome. Gain in accuracy was also observed for Minnesotans using the study-specific reference panel, although the increase was smaller than in Sardinians, especially for rare variants. Notably, a combined panel including both study-specific and 1000G reference panels improved imputation accuracy only in the Minnesota sample, and only at rare sites. Finally, we found that when imputation is performed with a study-specific reference panel, cutoffs different from the standard thresholds of MACH-Rsq and IMPUTE-INFO metrics should be used to efficiently filter badly imputed rare variants. This study thus provides general guidelines for researchers planning large-scale genetic studies. PMID:25293720

  10. 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

  11. Human parvovirus B19 infection and hydrops fetalis in Rio de Janeiro, Brazil.

    PubMed

    Cubel, R C; Garcia, A G; Pegado, C S; Ramos, H I; Fonseca, M E; Clewley, J P; Cohen, B J; Nascimento, J P

    1996-01-01

    Formalin-fixed paraffin embedded lung and liver tissue from 23 cases of non immune hydrops fetalis and five control cases, in which hydrops were due to syphilis (3) and genetic causes (2), were examined for the presence of human parvovirus B19 by DNA hybridisation. Using in situ hybridisation with a biotynilated probe one positive case was detected. Using 32P-labelled probes in a dot blot assay format, five further positives were obtained. These were all confirmed as positive by a nested polymerase chain reaction assay. Electron microscopy revealed virus in all these five positive cases. The six B19 DNA positive cases of hydrops fetalis were from 1974, 1980, 1982, 1987 and 1988, four of which occurred during the second half of the year, confirming the seasonality of the disease. PMID:8736082

  12. 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

  13. Clinical interpretation of antineutrophil cytoplasmic antibodies: parvovirus B19 infection as a pitfall

    PubMed Central

    Hermann, J; Demel, U; Stunzner, D; Daghofer, E; Tilz, G; Graninger, W

    2005-01-01

    Objective: To investigate whether positive ANCA test results may be a common feature of acute parvovirus B19 infection. Methods: Sera were analysed from 1242 patients from a rheumatology outpatient clinic for reactivity with parvovirus B19 and EBV antibodies. They were tested for the presence of PR3-ANCA and MPO-ANCA, along with sera known to contain IgM antibodies to these viruses obtained from among 41 366 samples submitted for virological screening. Results: ANCA were found in 10% (5/50) of the sera positive for IgM antibodies to parvovirus and in 3/51 sera containing IgM antibodies to EBV. Three of six patients with arthritis and concomitant parvovirus infection were found positive for PR3-ANCA and two were found positive for MPO-ANCA. All six patients tested negative for ANCA after six months of follow up. Conclusions: PR3-ANCA and MPO-ANCA may occur transiently in patients with acute B19 infection or infectious mononucleosis, highlighting the importance of repeated antibody tests in oligosymptomatic clinical conditions in which systemic autoimmune disease is suspected. PMID:15485998

  14. 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

  15. 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

    ... Procedures (44 FR 11034, February 26, 1979); and 3. Will not have a significant economic impact, positive or..., Inc. Model CL-600-2B19 (Regional Jet Series 100 & 440) Airplanes AGENCY: Federal Aviation.... Applicability (c) This AD applies to Bombardier, Inc. Model CL-600-2B19 (Regional Jet Series 100 &...

  16. 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

    ... 12866; 2. Is not a ``significant rule'' under the DOT Regulatory Policies and Procedures (44 FR 11034.... Model CL-600-2B19 (Regional Jet Series 100 & 440) Airplanes AGENCY: Federal Aviation Administration (FAA... Part 2, ``Airworthiness Limitations,'' of the Bombardier CL-600-2B19 Maintenance Requirements...

  17. 76 FR 29997 - Airworthiness Directives; Bombardier, Inc. Model CL-600-2B19 (Regional Jet Series 100 & 440...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-24

    ... . SUPPLEMENTARY INFORMATION: Airworthiness Directive 2011-07-06, amendment 39-16643 (76 FR 18024, April 1, 2011.... Model CL-600-2B19 (Regional Jet Series 100 & 440) Airplanes AGENCY: Federal Aviation Administration (FAA.... Model CL-600-2B19 (Regional Jet Series 100 & 440) airplanes. In the AD as published, the reference...

  18. 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. PMID:26984090

  19. Guillain-Barré Syndrome Associated with Primary Parvovirus B19 Infection in an HIV-1-Infected Patient

    PubMed Central

    Bucher Praz, Caroline; Dessimoz, Cédric; Bally, Frank; Reymond, Sitthided; Troillet, Nicolas

    2012-01-01

    Parvovirus B19 (B19V) infection has rarely been reported as responsible for Guillain-Barré syndrome (GBS). We present the case of a 63-year-old man with AIDS who presented with rapidly progressing weakness of his inferior limbs and a newly appeared pancytopenia. CSF examination and electromyography were characteristic for GBS. Very high CSF and serum B19V DNA concentrations were present, in the absence of IgG or IgM against B19V. The neurologic and hematologic abnormalities improved after a 5-day course of i.v. immunoglobulins in parallel with a dramatic decrease in the B19V viral load. PMID:23251163

  20. 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. PMID:23474534

  1. 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...

  2. 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...

  3. Genotype-dependent expression of specific members of potato protease inhibitor gene families in different tissues and in response to wounding and nematode infection.

    PubMed

    Turrà, David; Bellin, Diana; Lorito, Matteo; Gebhardt, Christiane

    2009-05-01

    Protease inhibitors (PIs) are small ubiquitous proteins with a variety of biological functions in plants, including protein stabilization, modulation of apoptosis and defense against pathogens. Kunitz-like inhibitors (PKPIs) and proteinase inhibitors 1 (PI-1) are abundant in storage organs of potato plants and are up-regulated in other tissues in response to biotic and abiotic stress. However, little information is available on genotype-dependent regulation of individual PKPI group- and PI-1 genes. We isolated, sequenced and characterized four novel full-length PI-1 cDNAs (PPI3A2, PPI3A4, PPI2C4 and PPI2C1A) from Solanum tuberosum cv. Desirée. Specific primers were developed for PI-1 genes PPI3A2, PPI3B2 and PPI2C4 and the three PKPI homology groups A, B and C. Their expression profiles were studied by semi-quantitative RT-PCR in comparison with transcripts of the PI-1, Pin2 and PR1 gene families in various tissues, after wounding and Globodera rostochiensis infection of nematode-resistant genotypes P40 and LB7/4/c-I-7, and susceptible cv. Desirée. Individual PI-1 genes and PKPI homology groups were expressed in a tissue- and genotype-dependent manner after wounding and nematode infection. The differences in PI expression patterns were related to the intensity, type of inhibitors produced, and the kinetics of induction. Therefore, different genotype-environment combinations produce different sets of PI transcripts. Potato plants reacted to G. rostochiensis infection by modulating PKPI, PI-1 and Pin2, but not PR1 gene expression, suggesting that the jasmonic acid but not the salicylic acid defense signaling pathway is activated. PI expression profiles were not correlated with the resistance status of the potato genotype infected with G. rostochiensis. PMID:19095329

  4. Development of a Novel Genus-Specific Real-Time PCR Assay for Detection and Differentiation of Bartonella Species and Genotypes

    PubMed Central

    Bai, Ying; Malania, Lile; Winchell, Jonas M.; Kosoy, Michael Y.

    2012-01-01

    The genus Bartonella includes numerous species with varied host associations, including several that infect humans. Development of a molecular diagnostic method capable of detecting the diverse repertoire of Bartonella species while maintaining genus specificity has been a challenge. We developed a novel real-time PCR assay targeting a 301-bp region of the ssrA gene of Bartonella and demonstrated specific amplification in over 30 Bartonella species, subspecies, and strains. Subsequent analysis of ssrA sequences was sufficient to discriminate Bartonella species and provided phylogenetic data consistent with that of gltA, a commonly used gene for differentiating Bartonella genotypes. Using this assay, we identified Bartonella DNA in 29% and 47% of blood specimens from elk in Wyoming and cattle in the Republic of Georgia, respectively. Sequence analysis of a subset of genotypes from elk specimens revealed a cluster most closely related to Bartonella capreoli, and genotypes from cattle were identified as Bartonella bovis, both Bartonella species commonly found in wild and domestic ruminants. Considering the widespread geographic distribution and infectivity potential to a variety of hosts, this assay may be an effective diagnostic method for identification of Bartonella infections in humans and have utility in Bartonella surveillance studies. PMID:22378904

  5. 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

  6. 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

  7. 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

  8. 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...

  9. Severe anemia due to parvovirus B19 in a silver haired boy.

    PubMed

    Verma, Nishant; Kumar, Archana; Kushwaha, Rashmi

    2016-01-01

    Griscelli syndrome (GS) is a rare autosomal recessive immunodeficiency disorder in which the affected children present with characteristic silvery-white hairs. The hair microscopy of these children is characteristic and is helpful in differentiating GS from Chediak-Higashi syndrome which also presents with immunodeficiency and silver hairs. We report a 17-month-old boy with GS type 2 who presented with severe anemia. Bone marrow examination of the child suggested parvovirus B19 as the cause of severe anemia, which was later confirmed by DNA polymerase chain reaction. PMID:26960654

  10. Adenovirus E1B 19-kilodalton protein overcomes the cytotoxicity of E1A proteins.

    PubMed Central

    White, E; Cipriani, R; Sabbatini, P; Denton, A

    1991-01-01

    Infection with adenovirus mutants carrying either point mutations or deletions in the coding region for the 19-kDa E1B gene product (19K protein) causes degradation of host cell and viral DNAs (deg phenotype) and enhanced cytopathic effect (cyt phenotype). Therefore, one function of the E1B 19K protein is to protect nuclear DNA integrity and preserve cytoplasmic architecture during productive adenovirus infection. When placed in the background of a virus incapable of expressing a functional E1A gene product, however, E1B 19K gene mutations do not result in the appearance of the cyt and deg phenotypes. This demonstrated that expression of the E1A proteins was responsible for inducing the appearance of the cyt and deg phenotypes. By constructing a panel of viruses possessing E1A mutations spanning each of the three E1A conserved regions in conjunction with E1B 19K gene mutations, we mapped the induction of the cyt and deg phenotypes to the amino-terminal region of E1A. Viruses that fail to express conserved region 3 (amino acids 140 to 185) and/or 2, (amino acids 121 to 185) or nonconserved sequences between conserved regions 2 and 1 of E1A (amino acids 86 to 120) were still capable of inducing cyt and deg. This indicated that activities associated with these regions, such as transactivation and binding to the product of the retinoblastoma susceptibility gene, were dispensable for induction of E1A-dependent cytotoxic effects. In contrast, deletion of sequences in the amino terminus of E1A (amino acids 22 to 107) resulted in extragenic suppression of the cyt and deg phenotypes. Therefore, a function affected by deletion of amino acids 22 to 86 of E1A is responsible for exerting cytotoxic effects in virally infected cells. Furthermore, transient high-level expression of the E1A region using a cytomegalovirus promoter plasmid expression vector was sufficient to induce the cyt and deg phenotypes, demonstrating that E1A expression alone is sufficient to exert these

  11. 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

  12. Physiological and genotype-specific factors associated with grain quality changes in rice exposed to high ozone.

    PubMed

    Jing, Liquan; Dombinov, Vitalij; Shen, Shibo; Wu, Yanzhen; Yang, Lianxin; Wang, Yunxia; Frei, Michael

    2016-03-01

    Rising tropospheric ozone concentrations in Asia affect the yield and quality of rice. This study investigated ozone-induced changes in rice grain quality in contrasting rice genotypes, and explored the associated physiological processes during the reproductive growth phase. The ozone sensitive variety Nipponbare and a breeding line (L81) containing two tolerance QTLs in Nipponbare background were exposed to 100 ppb ozone (8 h per day) or control conditions throughout their growth. Ozone affected grain chalkiness and protein concentration and composition. The percentage of chalky grains was significantly increased in Nipponbare but not in L81. Physiological measurements suggested that grain chalkiness was associated with a drop in foliar carbohydrate and nitrogen levels during grain filling, which was less pronounced in the tolerant L81. Grain total protein concentration was significantly increased in the ozone treatment, although the albumin fraction (water soluble protein) decreased. The increase in protein was more pronounced in L81, due to increases in the glutelin fraction in this genotype. Amino acids responded differently to the ozone treatment. Three essential amino acids (leucine, methionine and threonine) showed significant increases, while seven showed significant treatment by genotype interactions, mostly due to more positive responses in L81. The trend of increased grain protein was in contrast to foliar nitrogen levels, which were negatively affected by ozone. A negative correlation between grain protein and foliar nitrogen in ozone stress indicated that higher grain protein cannot be explained by a concentration effect in all tissues due to lower biomass production. Rather, ozone exposure affected the nitrogen distribution, as indicated by altered foliar activity of the enzymes involved in nitrogen metabolism, such as glutamine synthetase and glutamine-2-oxoglutarate aminotransferase. Our results demonstrate differential responses of grain quality

  13. 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

  14. [Aplastic crisis due to parvovirus B19 and Epstein-Barr virus in a patient with hereditary spherocytosis].

    PubMed

    Leoz Gordillo, I; Pérez Suárez, E

    2015-01-01

    Anemic syndrome in childhood requires a diagnosis and urgent treatment guided by systematic protocols that can avoid unnecessary additional testing. The case of a 4 year-old girl with fatigue and intermittent fever of 7 days duration, accompanied by abdominal pain is presented. She had regular general health status, with mucocutaneous jaundice, a grade III/VI/iv murmur, and painful abdomen with hepatosplenomegaly. The blood analysis showed a hypo-regenerative anemia with increased LDH and indirect bilirubin. The Coombs Test was negative, with spherocytes being observed in the peripheral blood smear. The IgM and IgG were positive for parvovirus B19 IgM and Epstein Barr virus, leading to the diagnosis of aplastic crisis in a patient with hereditary spherocytosis. No specific treatment was required. Under the suspicion of anemic syndrome in emergencies, the ABCDE sequence must be followed. Through the history, physical examination and basic laboratory tests, an initial diagnostic approach can be made. Specific etiological tests should be based on this first study. PMID:24629905

  15. Age-specific prevalence of HPV genotypes in cervical cytology samples with equivocal or low-grade lesions

    PubMed Central

    Brismar-Wendel, S; Froberg, M; Hjerpe, A; Andersson, S; Johansson, B

    2009-01-01

    Background: To define the spectrum of human papillomavirus (HPV) types and establish an age limit for triage HPV testing in atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL). Materials and methods: 343 liquid-based cytological samples from the population-based screening programme with minor abnormalities were subjected to HPV genotyping (Linear Array, Roche, Basel, Switzerland). Results: High-risk human papillomavirus (HR-HPV) was found in 71% of LSIL and 49% of ASCUS cases (P<0.001). High-risk human papillomavirus prevalence was age-dependent in LSIL (P=0.01), with decreasing prevalence until the age of 50 years, followed by a slight increase. Human papillomavirus type 16 was the most common HR-HPV, found in 23% of HPV-positive women. Human papillomavirus type 18 was the sixth most common, found in 9.9% (P<0.001). An age-dependent quadratic trend was observed for multiple infections (P=0.01) with a trough at about 42 years. The most common HR-HPV types to show a coinfection with HPV16 (clade 9) were HPV39 (28%), 45 (38%), and 59 (46%), belonging to HPV18 clade 7. The frequency of low-risk (LR) vs probable HR and HR-HPV also followed an age-dependent quadratic trend. Conclusions: After the age of 25 years, HR-HPV prevalence is similar in LSIL and ASCUS cases, motivating a low age limit for triage HPV testing. Multiple infections and LR/HR-HPV dominance are age-dependent. Genotyping in longitudinal design is needed to elucidate the importance of multiple infections in cancer progression and in cross-protection from vaccination. PMID:19623178

  16. Formation of empty B19 parvovirus capsids by the truncated minor capsid protein.

    PubMed Central

    Wong, S; Momoeda, M; Field, A; Kajigaya, S; Young, N S

    1994-01-01

    We previously reported that empty capsids of B19 parvovirus were formed by the major capsid protein (VP2) alone expressed in a baculovirus system, but the minor capsid protein (VP1), longer by 227 amino acids, alone did not form empty capsids. We report here further investigations of the constraints on capsid formation by truncated versions of VP1. Studies were performed with recombinant baculoviruses expressed in Sf9 cells. Severely shortened VP1, extended beyond the VP2 core sequence by about 70 amino acids of the unique region, formed capsids normal in appearance; longer versions of VP1 also formed capsids but did so progressively less efficiently and produced capsids of more markedly dysmorphic appearance as the VP1-unique region was lengthened. Images PMID:8207846

  17. A comparison of human papillomavirus genotype-specific DNA and E6/E7 mRNA detection to identify anal precancer among HIV-infected men who have sex with men

    PubMed Central

    Castle, Philip E.; Follansbee, Stephen; Borgonovo, Sylvia; Tokugawa, Diane; Schwartz, Lauren M.; Lorey, Thomas S.; LaMere, Brandon; Gage, Julia C.; Fetterman, Barbara; Darragh, Teresa M.; Rodriguez, Ana Cecilia; Wentzensen, Nicolas

    2012-01-01

    Background Human papillomavirus (HPV) RNA detection is reportedly more specific for the detection of anogenital precancer than HPV DNA but it is unknown whether this is due to detection of RNA or due to HPV genotype restriction. Materials and Methods 363 human immunodeficiency virus (HIV)-positive men who have sex with men had two anal cytology samples taken and were evaluated using high-resolution anoscopy and biopsies of visible lesions. Anal specimens were tested for E6/E7 RNA for 5 carcinogenic HPV genotypes (HPV16, 18, 31, 33, and 45) and tested for the DNA of 13 carcinogenic HPV genotypes. Results DNA testing was more likely to be positive than RNA testing (53% vs. 48%, p = 0.02) for the same 5 HPV genotypes in aggregate. When restricted to 5 HPV genotypes targeted by the RNA test, the sensitivity to detect anal precancer was the same for DNA and RNA (81%) while RNA was more specific than DNA (65% vs. 58%, p = 0.007). By comparison, DNA detection of all 13 carcinogenic HPV genotypes was more sensitive (96% vs. 81%, p = 0.001) but much less specific (65% vs. 33%, p < 0.001) compared to RNA detection of the 5 HPV genotypes. Conclusion After controlling for HPV genotypes, RNA was only slightly more specific than DNA detection for anal precancer. Impact DNA or RNA testing for a subset of the most carcinogenic HPV genotypes may be useful for distinguishing between those HPV-positive men at higher and lower risk of anal precancer and cancer. PMID:23155136

  18. Modulation of p53-mediated transcriptional repression and apoptosis by the adenovirus E1B 19K protein.

    PubMed Central

    Sabbatini, P; Chiou, S K; Rao, L; White, E

    1995-01-01

    BRK cell lines that stably express adenovirus E1A and a murine temperature-sensitive p53 undergo apoptosis when p53 assumes the wild-type conformation. Expression of the E1B 19,000-molecular-weight (19K) protein rescues cells from this p53-mediated apoptosis and diverts cells to a growth-arrested state. As p53 likely functions as a tumor suppressor by regulating transcription, the ability of the E1B 19K protein to regulate p53-mediated transactivation and transcriptional repression was investigated. In promoter-reporter assays the E1B 19K did not block p53-mediated transactivation but did alleviate p53-mediated transcriptional repression. E1B 19K expression permitted efficient transcriptional activation of the p21/WAF-1/cip-1 mRNA by p53, consistent with maintenance of the growth arrest function of p53. The E1B 19K protein is thereby unique among DNA virus-transforming proteins that target p53 for inactivation in that it selectively modulates the transcriptional properties of p53. The E1B 19K protein also rescued cells from apoptosis induced by inhibitors of transcription and protein synthesis. This suggests that cell death may result from the inhibition of expression of survival factors which function to maintain cell viability. p53 may induce apoptosis through generalized transcriptional repression. In turn, the E1B 19K protein may prevent p53-mediated apoptosis by alleviating p53-mediated transcriptional repression. PMID:7823921

  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. PMID:22900500

  20. A new pyrosequencing assay for rapid detection and genotyping of Shiga toxin, intimin and O157-specific rfbE genes of Escherichia coli.

    PubMed

    Goji, Noriko; Mathews, Amit; Huszczynski, George; Laing, Chad R; Gannon, Victor P J; Graham, Morag R; Amoako, Kingsley K

    2015-02-01

    Shiga toxin (stx)-producing Escherichia coli (STEC) contamination in food and water is one of the most recognized concerns and a major financial burden in human hygiene control worldwide. Rapid and highly reliable methods of detecting and identifying STEC causing gastroenteric illnesses are crucial to prevent foodborne outbreaks. A number of tests have been developed and commercialized to detect STEC using molecular microbiology techniques. Most of these are designed to identify virulence factors such as Shiga toxin and intimin as well as E. coli O and H antigen serotype specific genes. In order to screen pathogenic STEC without relying on O:H serotyping, we developed a rapid detection and genotyping assay for STEC virulence genes using a PCR-pyrosequencing application. We adapted the PyroMark Q24 Pyrosequencing platform for subtyping 4 major virulence genes, Shiga toxin 1 and 2 (stx1 and stx2), intimin (eae) and O157-antigen gene cluster target rfbE, using Single Nucleotide Polymorphism (SNP) analysis. A total of 224 E. coli strains including isolates from Canadian environment, food and clinical cases were examined. Based on the multiple alignment analysis of 30-80 base nucleotide pyrogram reads, three alleles of the Shiga toxin 1a gene (stx1a) (stx1a-I, stx1a-II, stx1a-III) were identified. Results of the stx1, stx2, eae and rfbE genotyping revealed that each group of O:H serotype shares distinctive characteristics that could be associated with the virulence of each genotype. O157:H7/NM carries stx1a-II (94%), stx2a (82%), λ/γ1-eae (100%) and rfbE type-H7/NM (100%). Whereas isolates of the "Top-6" serotypes (O26, O45, O103, O111, O121, O145) had a high incidence of stx1a-I (90%) and stx2a (100%). stx1a-III (60%) was only observed in non Top-7 (Top-6 plus O157) STEC and Shigella spp. The entire assay, from extracting DNA from colonies on a plate to the generation of sequence information, can be completed in 5h. The method of profiling these 4 STEC pathogenic

  1. Genotyping of Trypanosoma cruzi Sublineage in Human Samples from a North-East Argentina Area by Hybridization with DNA Probes and Specific Polymerase Chain Reaction (PCR)

    PubMed Central

    Diez, Cristina; Lorenz, Virginia; Ortiz, Silvia; Gonzalez, Verónica; Racca, Andrea; Bontempi, Iván; Manattini, Silvia; Solari, Aldo; Marcipar, Iván

    2010-01-01

    We have evaluated blood samples of chronic and congenital Trypanosoma cruzi-infected patients from the city of Reconquista located in the northeast of Argentina where no information was previously obtained about the genotype of infecting parasites. Fourteen samples of congenital and 19 chronical patients were analyzed by hybridization with DNA probes of minicircle hypervariable regions (mHVR). In congenital patients, 50% had single infections with TcIId, 7% single infections with TcIIe, 29% mixed infections with TcIId/e, and 7% had mixed infections with TcIId/b and 7% TcIId/b, respectively. In Chronical patients, 52% had single infections with TcIId, 11% single infections with TcIIe, 26% had mixed infections with TcIId/e, and 11% had non-identified genotypes. With these samples, we evaluated the minicircle lineage-specific polymerase chain reaction assay (MLS-PCR), which involves a nested PCR to HVR minicircle sequences and we found a correlation with hybridization probes of 96.4% for TcIId and 54.8% for TcIIe. PMID:20064998

  2. 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

  3. 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

  4. The gender-specific apolipoprotein E genotype influence on the distribution of plasma lipids and apolipoproteins in the population of Rochester, MN. III. Correlations and covariances

    SciTech Connect

    Reilly, S.L.; Sing, C.F.; Ferrell, R.E.

    1994-11-01

    The gender-specific influence that the apolipoprotein E (Apo E) polymorphism has on the correlations and covariances between pairs of nine plasma lipid and apolipoprotein traits (total cholesterol; ln triglycerides; high-density-lipoprotein cholesterol; apolipoproteins AI, AII, B, CII, lnCIII, and lnE) was studied in 507 unrelated individuals representative of the adult population of Rochester, MN. Analyses are presented separately for females and males. The Apo E polymorphism had a significant influence on a large number (10 of 36) of correlations and covariances in females and on a small number of (3 of 36) in males. The contribution of allelic variation in the Apo E gene to the definition of multivariate measures of the 36-dimensional correlation structure was evaluated. The influence of APo E genotype on correlation structure was gender dependent. These findings were used to demonstrate how heterogeneity of risk-factor correlations and covariances among genotype-gender subgroups of the population at large may influence the evaluation of risk of coronary artery disease. 73 refs., 8 figs., 4 tabs.

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

    PubMed Central

    2014-01-01

    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. PMID:24456189

  6. 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.

  7. Seizure and hepatosplenomegaly-rare manifestation of parvovirus B-19: a case report and review of the literature.

    PubMed

    Kamlesh, Yadav; Pallav, Gupta; Manjula, Murari; Rohan, Malik

    2011-01-01

    Parvovirus B19 is the etiologic agent of erythema infectiosum (fifth disease), a fever-rash illness occurring in childhood. We present a 10 month old child with high grade fever for 10 days, generalized tonic-clonic seizure, bilateral cervical lymphadenopathy, generalized maculopapular rash, hematemesis and malena. Bone marrow aspiration and liver biopsy were done. EBV serology and parvovirus PCR were also performed. Bone marrow aspiration and biopsy showed giant pro-erythroblast consistent with parvovirus infection. PCR showed amplification of parvovirus genomic sequences. Present case highlights an atypical presentation of Parvovirus B19 infection as fever, rash and hepatosplenomegaly. PMID:21760806

  8. Effects of Parvovirus B19 Infection in Renal Transplant Recipients: A Retrospective Review of Three Cases.

    PubMed

    Krishnan, Prathik; Ramadas, Poornima; Rajendran, Prejith P; Madhavan, Parvathy; Alex, Asha; Jayaschandran, Vivek; Humayun, Shaesta G; Ali, Nicole; Sachdeva, Mala; Flecha, Antonette; Basu, Amit; Bhaskaran, Madhu; Molmenti, Ernesto P

    2015-06-01

    Parvovirus B19 (PVB19) is a DNA virus which causes clinically relevant infection in renal transplant recipients (RTR) leading to significant morbidity. Manifestations include erythropoietin resistant anemia, proteinuria, and glomerulosclerosis in the allograft. Severe infection may require administration of intravenous immunoglobulin, reduction in immunosuppression and transfusions. The major challenge in managing and preventing the infection in RTR involves the act of balancing the decreased level of immunosuppression and the risk of rejection. The objective of this article is to understand the importance of PVB19 infection and its outcome in RTR. We reviewed the medical records of three RTR with confirmed PVB19 infection and recorded patient information including demographics, clinical and laboratory data, management, and outcome. The average time of occurrence of PVB19 infection as transplant was 8.6 weeks and they presented with symptomatic anemia. Elevated creatinine values were noted in two of them. Following treatment, anemia improved and creatinine values returned to baseline. One of them developed an early relapse and had to be treated once again similarly. We emphasize the importance of maintaining a high index of suspicion for PVB19 infection in patients with anemia in the posttransplant phase, especially in patients on higher doses of immunosuppressants. Early and proper treatment can prevent worsening clinical condition and possible effects on the allograft. PMID:26060378

  9. Modest truncation of the major capsid protein abrogates B19 parvovirus capsid formation.

    PubMed Central

    Kawase, M; Momoeda, M; Young, N S; Kajigaya, S

    1995-01-01

    In vitro studies have suggested an important role for the minor capsid protein (VP1) unique region and the junction between VP1 and the major capsid protein (VP2) in the neutralizing immune response to B19 parvovirus. We investigated the role of the NH2-terminal region of the major structural protein in capsid structure by expressing progressively more truncated versions of the VP2 gene followed by analysis using immunoblotting and electron microscopy of density gradient-purified particles. Deletion of the first 25 amino acids (aa) of VP2 did not affect capsid assembly. Altered VP2 with truncations to aa 26 to 30, including a single amino acid deletion at position 25, failed to self-assemble but did participate with normal VP2 in the capsid structure. The altered region corresponds to the beginning of the beta A antiparallel strand. Truncations beyond aa 30 were incompatible with either self-assembly or coassembly, probably because of deletion of the beta B strand, which helps to form the core structure of the virus. PMID:7666560

  10. Genotype-specific effects of Mecp2 loss-of-function on morphology of Layer V pyramidal neurons in heterozygous female Rett syndrome model mice

    PubMed Central

    Rietveld, Leslie; Stuss, David P.; McPhee, David; Delaney, Kerry R.

    2015-01-01

    Rett syndrome (RTT) is a progressive neurological disorder primarily caused by mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2). The heterozygous female brain consists of mosaic of neurons containing both wild-type MeCP2 (MeCP2+) and mutant MeCP2 (MeCP2-). Three-dimensional morphological analysis was performed on individually genotyped layer V pyramidal neurons in the primary motor cortex of heterozygous (Mecp2+/-) and wild-type (Mecp2+/+) female mice ( > 6 mo.) from the Mecp2tm1.1Jae line. Comparing basal dendrite morphology, soma and nuclear size of MeCP2+ to MeCP2- neurons reveals a significant cell autonomous, genotype specific effect of Mecp2. MeCP2- neurons have 15% less total basal dendritic length, predominantly in the region 70–130 μm from the cell body and on average three fewer branch points, specifically loss in the second and third branch orders. Soma and nuclear areas of neurons of mice were analyzed across a range of ages (5–21 mo.) and X-chromosome inactivation (XCI) ratios (12–56%). On average, MeCP2- somata and nuclei were 15 and 13% smaller than MeCP2+ neurons respectively. In most respects branching morphology of neurons in wild-type brains (MeCP2 WT) was not distinguishable from MeCP2+ but somata and nuclei of MeCP2 WT neurons were larger than those of MeCP2+ neurons. These data reveal cell autonomous effects of Mecp2 mutation on dendritic morphology, but also suggest non-cell autonomous effects with respect to cell size. MeCP2+ and MeCP2- neuron sizes were not correlated with age, but were correlated with XCI ratio. Unexpectedly the MeCP2- neurons were smallest in brains where the XCI ratio was highly skewed toward MeCP2+, i.e., wild-type. This raises the possibility of cell non-autonomous effects that act through mechanisms other than globally secreted factors; perhaps competition for synaptic connections influences cell size and morphology in the genotypically mosaic brain of RTT model mice. PMID:25941473

  11. Age-specific prevalence of HPV16/18 genotypes in cervical cancer: A systematic review and meta-analysis.

    PubMed

    Hammer, Anne; Rositch, Anne; Qeadan, Fares; Gravitt, Patti E; Blaakaer, Jan

    2016-06-15

    The prevalence of HPV16/18 in cervical cancer has been reported to decline with age in some papers. However, whether this decline in proportion of cancers positive for HPV16/18 is consistently observed across studies remains to be elucidated. Thus, the aim of this study was to identify papers reporting data on age-specific prevalence of HPV16/18 in cervical cancer and to summarize the results. We employed MEDLINE and Embase for a systematic literature search and thereby identified a total of 644 papers published in the period 1999-2015, of which 15 papers, reporting cross-sectional data, were included for review (11,526 cervical cancers). The prevalence of HPV16/18 in cervical cancer declined significantly with age (ρ = -0.83, p = 0.04) from 74.8% (95% CI 67.6-80.8) in women aged 30-39 years to 56.8% (95% CI 43.9-68.8) in women aged ≥70 years. As the HPV16/18 positive cancers are prevented in fully vaccinated cohorts, the age-specific epidemiology of cervical cancer is anticipated to change, with a shift in peak incidence rate to older ages. It will be important for integrated vaccination and screening strategies to consider predicted change in the age-specific epidemiology of cervical cancer. PMID:26661889

  12. 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

    ... rule'' under the DOT Regulatory Policies and Procedures (44 FR 11034, February 26, 1979); and 3. Will.... Model CL-600-2B19 (Regional Jet Series 100 & 440) Airplanes; Model CL-600-2C10 (Regional Jet Series 700, 701, & 702) Airplanes; Model CL-600-2D15 (Regional Jet Series 705) Airplanes; and Model...

  13. 75 FR 59073 - Airworthiness Directives; Bombardier, Inc. Model CL-600-2B19 (Regional Jet Series 100 & 440...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-27

    ... to the specified products. That NPRM was published in the Federal Register on April 8, 2010 (75 FR... Order 12866; 2. Is not a ``significant rule'' under the DOT Regulatory Policies and Procedures (44 FR.... Model CL-600-2B19 (Regional Jet Series 100 & 440) Airplanes; Model CL-600-2C10 (Regional Jet Series...

  14. 75 FR 60604 - Airworthiness Directives; Bombardier, Inc. Model CL-600-2B19 (Regional Jet Series 100 & 440...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-01

    ... (75 FR 34657). That NPRM proposed to correct an unsafe condition for the specified products. The MCAI...'' under the DOT Regulatory Policies and Procedures (44 FR 11034, February 26, 1979); and 3. Will not have.... Model CL-600-2B19 (Regional Jet Series 100 & 440) Airplanes; Model CL-600-2C10 (Regional Jet Series...

  15. 75 FR 31324 - Airworthiness Directives; Bombardier, Inc. Model CL-600-2B19 (Regional Jet Series 100 & 440...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-03

    ... ``significant rule'' under the DOT Regulatory Policies and Procedures (44 FR 11034, February 26, 1979); and 3... in the AD docket. Relevant Service Information Bombardier has issued Temporary Revision (TR) 2A-43...-600-2B19 Maintenance Requirements Manual; and Canadair Regional Jet TR RJ/178-1, dated March 8,...

  16. 76 FR 53046 - Airworthiness Directives; Bombardier, Inc. Model CL-600-2B19 (Regional Jet Series 100 & 440...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-25

    ... Regulatory Policies and Procedures (44 FR 11034, February 26, 1979); and 3. Will not have a significant.... Model CL-600-2B19 (Regional Jet Series 100 & 440) Airplanes AGENCY: Federal Aviation Administration (FAA... generator (IDG) power cables. Additionally, the hydraulic line support brackets located at the...

  17. 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

    ... 12866; 2. Is not a ``significant rule'' under the DOT Regulatory Policies and Procedures (44 FR 11034.... Model CL-600-2B19 (Regional Jet Series 100 & 440) Airplanes AGENCY: Federal Aviation Administration (FAA... of on-ground hydraulic accumulator screw cap/end cap failure have been experienced on...

  18. 75 FR 63054 - Airworthiness Directives; Bombardier, Inc. Model CL-600-2B19 (Regional Jet Series 100 & 440...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-14

    ... published in the Federal Register on January 5, 2010 (75 FR 258). That NPRM proposed to correct an unsafe...'' under the DOT Regulatory Policies and Procedures (44 FR 11034, February 26, 1979); and 3. Will not have.... Model CL-600-2B19 (Regional Jet Series 100 & 440) Airplanes AGENCY: Federal Aviation Administration...

  19. Small RNA and degradome deep sequencing reveals drought-and tissue-specific micrornas and their important roles in drought-sensitive and drought-tolerant tomato genotypes.

    PubMed

    Candar-Cakir, Bilgin; Arican, Ercan; Zhang, Baohong

    2016-08-01

    Drought stress has adverse impacts on plant production and productivity. MicroRNAs (miRNAs) are one class of noncoding RNAs regulating gene expression post-transcriptionally. In this study, we employed small RNA and degradome sequencing to systematically investigate the tissue-specific miRNAs responsible to drought stress, which are understudied in tomato. For this purpose, root and upground tissues of two different drought-responsive tomato genotypes (Lycopersicon esculentum as sensitive and L. esculentum var. cerasiforme as tolerant) were subjected to stress with 5% polyethylene glycol for 7 days. A total of 699 conserved miRNAs belonging to 578 families were determined and 688 miRNAs were significantly differentially expressed between different treatments, tissues and genotypes. Using degradome sequencing, 44 target genes were identified associated with 36 miRNA families. Drought-related miRNAs and their targets were enriched functionally by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Totally, 53 miRNAs targeted 23 key drought stress- and tissue development-related genes, including DRP (dehydration-responsive protein), GTs (glycosyltransferases), ERF (ethylene responsive factor), PSII (photosystem II) protein, HD-ZIP (homeodomain-leucine zipper), MYB and NAC-domain transcription factors. miR160, miR165, miR166, miR171, miR398, miR408, miR827, miR9472, miR9476 and miR9552 were the key miRNAs functioning in regulation of these genes and involving in tomato response to drought stress. Additionally, plant hormone signal transduction pathway genes were differentially regulated by miR169, miR172, miR393, miR5641, miR5658 and miR7997 in both tissues of both sensitive and tolerant genotypes. These results provide new insight into the regulatory role of miRNAs in drought response with plant hormone signal transduction and drought-tolerant tomato breeding. PMID:26857916

  20. 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

  1. 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

  2. Hepatitis C virus-specific T-cell response correlates with hepatitis activity and donor IL28B genotype early after liver transplantation.

    PubMed

    Tsuzaki, Ryuichiro; Takaki, Akinobu; Yagi, Takahito; Ikeda, Fusao; Koike, Kazuko; Iwasaki, Yoshiaki; Shiraha, Hidenori; Miyake, Yasuhiro; Sadamori, Hiroshi; Shinoura, Susumu; Umeda, Yuzo; Yoshida, Ryuichi; Nobuoka, Daisuke; Utsumi, Masashi; Nakayama, Eiichi; Fujiwara, Toshiyoshi; Yamamoto, Kazuhide

    2014-01-01

    It is not known how the immune system targets hepatitis C virus (HCV)-infected HLA-mismatched hepatocytes under immune-suppressed conditions after orthotopic liver transplantation (OLT). In addition, the relationship between the HCV-specific immune response and IL28B variants as predictors of HCV clearance has not been well-characterized. We determined the IL28B polymorphisms for 57 post-OLT HCV carriers, and we assessed the HCV-specific immune responses by measuring the peripheral blood mononuclear cell-derived HCV-specific interferon-gamma (IFN-γ) response using an enzyme-linked immunospot assay. At 1-3 years after OLT, patients with no active hepatitis showed higher total spots on the immunospot assay. At>3 years after OLT, patients with resolved HCV showed higher levels of core, NS3, NS5A, and total spots compared to the chronic hepatitis patients. The IL28B major genotype in the donors correlated with higher spot counts for NS5A and NS5B proteins at 1-3 years after OLT. In the post-OLT setting, the HCV-specific immune response could be strongly induced in patients with no active hepatitis with an IL28B major donor or sustained virological response. Strong immune responses in the patients with no active hepatitis could only be maintained for 3 years and diminished later. It may be beneficial to administer IFN treatment starting 3 years after OLT, to induce the maximum immunological effect. PMID:25338486

  3. 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...

  4. A genotype-specific, randomized controlled behavioral intervention to improve the neuroemotional outcome of cardiac surgery: study protocol for a randomized controlled trial

    PubMed Central

    2013-01-01

    Background Cardiac surgery is one of the most commonly performed surgical procedures worldwide with >700,000 surgeries in 2006 in the US alone. Cardiac surgery results in a considerable exposure to physical and emotional stress; stress-related disorders such as depression or post-traumatic stress disorder are the most common adverse outcomes of cardiac surgery, seen in up to 20% of patients. Using information from a genome-wide association study to characterize genetic effects on emotional memory, we recently identified a single nucleotide polymorphism of the glucocorticoid receptor gene (the Bcll single nucleotide polymorphism) as a significant genetic risk factor for traumatic memories from cardiac surgery and symptoms of post-traumaticstress disorder. The Bcll high-risk genotype (Bcll GG) has a prevalence of 16.6% in patients undergoing cardiac surgery and is associated with increased glucocorticoid receptor signaling under stress. Concomitant animal experiments have confirmed an essential role of glucocorticoid receptor activation for traumatic memory formation during stressful experiences. Early cognitive behavioral intervention has been shown to prevent stress-related disorders after heart surgery. Methods/Design The proposed study protocol is based on the above mentioned earlier findings from animal experiments and preclinical studies in volunteers. Patients (n = 872) will be genotyped for the Bcll single nucleotide polymorphism before surgery, which should result in 120 homozygous high-risk carriers of the Bcll GG allele and 240 randomly selected low-risk heterozygous or non-carriers of the single nucleotide polymorphism. All patients will then undergo randomization to either cognitive behavioral intervention or a control intervention consisting of non-specific general information about the role of stress in heart disease. The primary efficacy endpoint will be post-traumatic stress levels at one year after surgery as determined by a standardized

  5. 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.

  6. 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. PMID:26688295

  7. A False Positive Dengue Fever Rapid Diagnostic Test Result in a Case of Acute Parvovirus B19 Infection.

    PubMed

    Izumida, Toshihide; Sakata, Hidenao; Nakamura, Masahiko; Hayashibara, Yumiko; Inasaki, Noriko; Inahata, Ryo; Hasegawa, Sumiyo; Takizawa, Takenori; Kaya, Hiroyasu

    2016-01-01

    An outbreak of dengue fever occurred in Japan in August 2014. We herein report the case of a 63-year-old man who presented with a persistent fever in September 2014. Acute parvovirus B19 infection led to a false positive finding of dengue fever on a rapid diagnostic test (Panbio Dengue Duo Cassette(TM)). To the best of our knowledge, there are no previous reports of a false positive result for dengue IgM with the dengue rapid diagnostic test. We believe that epidemiological information on the prevalence of parvovirus B19 is useful for guiding the interpretation of a positive result with the dengue rapid diagnostic test. PMID:27181552

  8. 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

  9. [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

  10. A cytotoxic nonstructural protein, NS1, of human parvovirus B19 induces activation of interleukin-6 gene expression.

    PubMed Central

    Moffatt, S; Tanaka, N; Tada, K; Nose, M; Nakamura, M; Muraoka, O; Hirano, T; Sugamura, K

    1996-01-01

    We examined the biological function of a nonstructural regulatory protein, NS1, of human parvovirus B19. Because of the cytotoxic activity of NS1, human hematopoietic cell lines, K562, Raji, and THP-1, were established as transfectants which produce the viral NS1 protein upon induction by using bacterial lactose repressor/operator system. NS1 was significantly produced in the three transfectant cells in an inducer dose- and time-dependent manner. Surprisingly, these three transfectants secreted an inflammatory cytokine, interleukin-6 (IL-6), in response to induction. However, no production of other related cytokines, IL-1beta, IL-8, or tumor necrosis factor alpha, was seen. Moreover, NS1-primed IL-6 induction was transiently demonstrated in primary human endothelial cells. Analysis with luciferase reporter plasmids carrying IL-6 promoter mutant fragments demonstrated that NS1 effect is mediated by a NF-kappaB binding site in the IL-6 promoter region, strongly implying that NS1 functions as a trans-acting transcriptional activator on the IL-6 promoter. Our novel finding, IL-6 induction by NS1, supports the possible relationship between parvovirus B19 infection and polyclonal activation of B cells in rheumatoid arthritis and indicates that NS1 protein may play a significant role in the pathogenesis of some B19-associated diseases by modulating the expression of host cellular genes. PMID:8970971

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

    DOE PAGESBeta

    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

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

    NASA Astrophysics Data System (ADS)

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

    2012-11-01

    Deformation of a B19' martensitic, polycrystalline Ni49.9Ti50.1 (at. %) shape memory alloy 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 situ neutron 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 dislocation 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.

  13. Genotype × genotype interactions between the toxic cyanobacterium Microcystis and its grazer, the waterflea Daphnia

    PubMed Central

    Lemaire, Veerle; Brusciotti, Silvia; van Gremberghe, Ineke; Vyverman, Wim; Vanoverbeke, Joost; De Meester, Luc

    2012-01-01

    Toxic algal blooms are an important problem worldwide. The literature on toxic cyanobacteria blooms in inland waters reports widely divergent results on whether zooplankton can control cyanobacteria blooms or cyanobacteria suppress zooplankton by their toxins. Here we test whether this may be due to genotype × genotype interactions, in which interactions between the large-bodied and efficient grazer Daphnia and the widespread cyanobacterium Microcystis are not only dependent on Microcystis strain or Daphnia genotype but are specific to genotype × genotype combinations. We show that genotype × genotype interactions are important in explaining mortality in short-time exposures of Daphnia to Microcystis. These genotype × genotype interactions may result in local coadaptation and a geographic mosaic of coevolution. Genotype × genotype interactions can explain why the literature on zooplankton–cyanobacteria interactions is seemingly inconsistent, and provide hope that zooplankton can contribute to the suppression of cyanobacteria blooms in restoration projects. PMID:25568039

  14. 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

  15. The association between MTHFR 677C>T genotype and folate status and genomic and gene-specific DNA methylation in the colon of individuals without colorectal neoplasia1234

    PubMed Central

    Hanks, Joanna; Ayed, Iyeman; Kukreja, Neil; Rogers, Chris; Harris, Jessica; Gheorghiu, Alina; Liu, Chee Ling; Emery, Peter

    2013-01-01

    Background: Decreased genomic and increased gene-specific DNA methylation predispose to colorectal cancer. Dietary folate intake and the methylenetetrahydrofolate reductase polymorphism (MTHFR 677C>T) may influence risk by modifying DNA methylation. Objective: We investigated the associations between MTHFR 677C>T genotype, folate status, and DNA methylation in the colon. Design: We conducted a cross-sectional study of 336 men and women (age 19–92 y) in the United Kingdom without colorectal neoplasia. We obtained blood samples for measurement of serum and red blood cell folate, plasma homocysteine, and MTHFR 677C>T genotype and colonic tissue biopsies for measurement of colonic tissue folate and DNA methylation (genomic- and gene-specific, estrogen receptor 1, ESR1; myoblast determination protein 1, MYOD1; insulin-like growth factor II, IGF2; tumor suppressor candidate 33, N33; adenomatous polyposis coli, APC; mut-L homolog 1, MLH1; and O6-methylguanine-DNA methyltransferase, MGMT) by liquid chromatography/electrospray ionization mass spectrometry and pyrosequencing, respectively. Results: Of the 336 subjects recruited, 185 (55%) carried the CC, 119 (35%) the CT, and 32 (10%) the TT alleles. No significant differences in systemic markers of folate status and colonic tissue folate between genotypes were found. The MTHFR TT genotype was not associated with genomic or gene-specific DNA methylation. Biomarkers of folate status were not associated with genomic DNA methylation. Relations between biomarkers of folate status and gene-specific methylation were inconsistent. However, low serum folate was associated with high MGMT methylation (P = 0.001). Conclusion: MTHFR 677C>T genotype and folate status were generally not associated with DNA methylation in the colon of a folate-replete population without neoplasia. This trial was registered at clinicaltrials.gov as ISRCTN43577261. PMID:24108782

  16. 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

  17. Parvovirus B19 infection presenting as pre-B-cell acute lymphoblastic leukemia: a transient and progressive course in two children.

    PubMed

    Yetgin, Sevgi; Cetin, Mualla; Aslan, Deniz; Ozyurek, Emel; Oyürek, Emel; Anlar, Banu; Uçkan, Duygu

    2004-10-01

    Parvovirus B19 is the causative agent of various forms of hematologic diseases such as aplastic crisis in patients with hemolytic anemia, aplastic anemia, hypoplastic anemia, and idiopathic thrombocytopenic purpura. In addition, parvovirus B19 infection may precede or be associated with acute lymphoblastic leukemia (ALL). The authors present two cases of parvovirus B19 infection and bone marrow infiltration with pre-B-cell lymphoblasts; one patients was diagnosed as having ALL, and the other patient, with neurologic findings, showed total resolution of the blastic morphology and phenotype. PMID:15454845

  18. Calpastatin and µ-calpain differ in their control of genotype specific residual variance of beef tenderness in Angus and MARC III steers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genotype variant effects of calpastatin (CAST) and µ-calpain (CAPN1) on mean beef tenderness have been widely characterized. We have tested whether these genetic variants also control residual (non-genetic) variation, and subsequently total phenotypic variation, of tenderness. Observation of rare ...

  19. 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. PMID:26010095

  20. 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

  1. A GC-box motif upstream of the B19 parvovirus unique promoter is important for in vitro transcription.

    PubMed Central

    Blundell, M C; Astell, C R

    1989-01-01

    Nucleotides upstream of the B19 parvovirus P6 promoter affect in vitro transcription in HeLa cell nuclear extracts. Comparison of the relative transcriptional strengths of equimolar mixes of plasmids containing the intact upstream sequence and plasmids containing deletions within these nucleotides identified several regions that affect transcription in vitro. A fragment containing two of five GC-box motifs which correspond to high-affinity SP1-binding sites was shown, by using a gel shift assay, to bind a HeLa cell factor (or factors). DNase I, methylation interference, and methylation protection footprinting demonstrated that the HeLa cell factor(s) bound to one of the two GC-box motifs within this fragment. Mutation of this GC box abolished factor binding and significantly reduces in vitro transcription from the P6 promoter. These results suggest that the B19 parvovirus promoter includes a complex regulatory region containing multiple sequences which affect promoter strength and that the GC-box motif is a major controlling sequence for in vitro transcription. Images PMID:2795719

  2. Neutralizing linear epitopes of B19 parvovirus cluster in the VP1 unique and VP1-VP2 junction regions.

    PubMed Central

    Saikawa, T; Anderson, S; Momoeda, M; Kajigaya, S; Young, N S

    1993-01-01

    Presentation of linear epitopes of the B19 parvovirus capsid proteins as peptides might be a useful vaccine strategy. We produced overlapping fusion proteins to span the viral capsid sequence, inoculated rabbits, and determined whether the resulting antisera contained antibodies that neutralized the ability of the virus to infect human erythroid progenitor cells. Antibodies that bound to virus in an enzyme-linked immunosorbent assay were present in antisera raised against 10 of 11 peptides; strongest activity was found for antisera against the carboxyl-terminal half of the major capsid protein. However, strong neutralizing activity was elicited in animals immunized with peptides from the amino-terminal portion of the unique region of the minor capsid protein and peptides containing the sequence of the junction region between the minor and major capsid proteins. The development of neutralizing activity in animals was elicited most rapidly with the fusion peptide from the first quarter of the unique region. A 20-amino-acid region of the unique region of the minor capsid protein was shown to contain a neutralizing epitope. Multiple antigenic peptides, based on the sequence of the unique region and produced by covalent linkage through a polylysine backbone, elicited strong neutralizing antibody responses. Synthetic peptides and fusion proteins containing small regions of the unique portion of the minor capsid protein might be useful as immunogens in a human vaccine against B19 parvovirus. Images PMID:7684458

  3. Genotyping methods.

    PubMed

    Tümmler, Burkhard

    2014-01-01

    Genotyping allows for the identification of bacterial isolates to the strain level and provides basic information about the evolutionary biology, population biology, taxonomy, ecology, and genetics of bacteria. Depending on the underlying question and available resources, Pseudomonas aeruginosa strains may be typed by anonymous fingerprinting techniques or electronically portable sequence-based typing methods such as multiple locus variable number tandem repeat (VNTR) analysis (MLVA), multilocus sequence typing, or oligonucleotide microarray. Macrorestriction fragment pattern analysis is a genotyping method that is globally applicable to all bacteria and hence has been and still is the reference method for strain typing in bacteriology. Agarose-embedded chromosomal DNA is cleaved with a rare-cutting restriction endonuclease and the generated 20-70 fragments are then separated by pulsed-field gel electrophoresis. The chapter provides a detailed step-by-step manual for SpeI genome fingerprinting of Pseudomonas chromosomes that has been optimized for SpeI fragment pattern analysis of P. aeruginosa. PMID:24818895

  4. 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

  5. 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

  6. 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.

  7. 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

  8. Mu (μ) Opioid Receptor Regulation of Ethanol-Induced Dopamine Response in the Ventral Striatum: Evidence of Genotype Specific Sexual Dimorphic Epistasis

    PubMed Central

    Job, Martin O.; Tang, Amanda; Hall, F. Scott; Sora, Ichiro; Uhl, George R.; Bergeson, Susan E.; Gonzales, Rueben A.

    2011-01-01

    Background Ethanol stimulates the dopaminergic mesoaccumbal pathway, which is thought to play a role in ethanol reinforcement. Mu (μ)-opioid (MOP) receptors modulate accumbal dopamine activity, but it is not clear whether MOP receptors are involved in the mechanism of ethanol-stimulated accumbal dopamine release. Methods We investigated the role that MOP receptors play in ethanol (2.0 g/kg)-stimulated accumbal dopamine release by using MOP receptor knockout mice (C57BL/6J-129SvEv and congenic C57BL/6J genotypes) along with blockade of MOP receptors with a μ1 selective antagonist (naloxonazine). Results Both gene deletion and pharmacological antagonism of the MOP receptor decreased ethanol-stimulated accumbal dopamine release compared with controls with female mice showing a larger effect in the C57BL/6J-129SvEv genotype. However, both male and female mice showed reduced ethanol-stimulated dopamine release in the congenic MOP receptor knockout mice (C57BL/6J). No differences in the time course of dialysate ethanol concentration were found in any of the experiments. Conclusions The data demonstrate the existence of a novel interaction between genotype and sex in the regulation of ethanol-stimulated mesolimbic dopamine release by the MOP receptor. This implies that a more complete understanding of the epistatic influences on the MOP receptor and mesolimbic dopamine function may provide more effective pharmacotherapeutic interventions in the treatment of alcoholism. PMID:17336938

  9. 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

  10. 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

  11. Stem cell transplantation in 6 children with parvovirus B19- induced severe aplastic anaemia or myelodysplastic syndrome.

    PubMed

    Urban, C; Lackner, H; Müller, E; Benesch, M; Strenger, V; Sovinz, P; Schwinger, W

    2011-11-01

    Parvovirus B19 (PVB19) induced severe aplastic anaemia (SAA) or myelodysplastic syndrome (MDS) is rare, and haematopoietic stem cell transplantation (HSCT) in this condition has not been reported so far. 6 children with SAA (n=4) or MDS (n=2) caused by acute PVB19 infection underwent HSCT under the protection of intravenous immunoglobulines. The 4 children with SAA received matched HLA bone marrow from a sibling (n=3) or peripheral unrelated blood stem cells (n=1). 1 patient had delayed erythrocyte engraftment, whereas 3 patients had an uneventful transplantation course. HSCT in one of the 2 children with MDS was complicated by poor graft function, the other patient engrafted without complications. In conclusion, HSCT in children with PVB19 induced SAA or MDS is feasible, even though some patients may develop delayed engraftment or prolonged poor graft function. PMID:22052631

  12. Successful treatment of severe aplastic anemia associated with human parvovirus B19 and Epstein-Barr virus in a healthy subject with allo-BMT.

    PubMed

    Kaptan, K; Beyan, C; Ural, A U; Ustün, C; Cetin, T; Avcu, F; Kubar, A; Aliş, M; Yalçin, A

    2001-08-01

    Several reports have noted pancytopenia associated with Human parvovirus B19 (PVB19) or Ebstein-Barr virus (EBV) infections in patients who have no history of immunodeficiency. To our knowledge, we report the first case of severe aplastic anemia associated with both EBV and PVB19 infections in a previously healthy 22-year-old man. He was admitted to our hematology service due to anemia and thrombocytopenia. He had no symptoms or signs of infections of these viruses. His bone marrow biopsy revealed a hypocellular marrow. Specific IgM and IgG antibodies to EBV and PVB19 were elevated. EBV and PVB19 virus genomes were detected by PCR in the bone marrow nucleated cells and the peripheral blood lymphocytes. Two months after treatment with prednisone, acyclovir, and intravenous immune globulin (IVIg), the genomes of both these viruses disappeared. However, his transfusion requirement for platelet suspensions and packed red blood cells persisted. The patient underwent allogeneic bone marrow transplant (allo-BMT) and has had an enduring complete hematological response for 8 months. PMID:11443638

  13. 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

  14. herg1b expression as a potential specific marker in pediatric acute myeloid leukemia patients with HERG 897K/K genotype.

    PubMed

    Erdem, Merve; Tekiner, Tugce Ayca; Fejzullahu, Arta; Akan, Gokce; Anak, Sema; Saribeyoglu, Ebru Tugrul; Ozbek, Ugur; Atalar, Fatmahan

    2015-04-01

    Human ether-a-go-go related gene (herg) encoding HERG K(+) channel has been demonstrated in many previous studies with its association to cell cycle progression and growth in tumor cells. The upregulated expression of HERG K+ channels was determined in different tumor types. Furthermore, not only full-length transcript herg1 but also a truncated isoform herg1b was shown to be expressed in cancer cells. In this study, the expression levels of herg1 and herg1b and the impact of K897T mutation on their expressions were investigated in pediatric acute myeloid leukemia (pAML). Expression levels of herg1 and herg1b isoforms were analyzed by quantitative real time polymerase chain reaction (PCR) in pAML patients together with healthy donors, and their expressions were confirmed by western blotting. The 2690 A>C nucleotide variation in KCNH2 gene corresponding to K897T amino acid change was analyzed by PCR followed by restriction enzyme digestion. herg1b overexpression was observed in tumor cells compared to healthy controls (P = .0024). However, herg1 expression was higher in healthy control cells than tumor cells (P = .001). The prevalence of polymorphic allele 897T was 26% in our patient group and 897T carriers showed increased herg1b expression compared to wild-type allele carriers. Our results demonstrate the presence of the increased levels of herg1b expression in pAML. In addition, we report for the first time that, pAML subgroup with HERG 897K/K genotype compared to 897K/T and T/T genotypes express increased levels of herg1b. In conclusion, HERG 897K/K genotype with increased level of herg1b expression might well be a prognostic marker for pAML. PMID:25247487

  15. 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

  16. 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

  17. First-principle simulation on the crystallization tendency and enhanced magnetization of Fe76B19P5 amorphous alloy

    NASA Astrophysics Data System (ADS)

    Wang, Yaocen; Zhang, Yan; Takeuchi, Akira; Makino, Akihiro; Liang, Yunye; Kawazoe, Yoshiyuki

    2015-01-01

    Iron-based amorphous alloys have attracted a growing interest due to their potential in the application of magnetic coil production. However, the magnetization of this kind of material is usually low due to the lack of long range ordering and high alloying element content. In this paper, an Fe76B19P5 amorphous alloy was simulated with ab initio molecular dynamics based on a previous simulation work on an Fe76Si9B10P5 amorphous alloy exhibiting that electron absorbers such as B and P can help enhance the magnetization of nearby Fe atoms. The present simulation results show that replacing Si with B can destabilize the amorphous structure, making it easier to crystallize, but no separate α-Fe participation can be observed in experiments during annealing due to its high B/P content. The results also show an increase in saturation magnetization by 8% can be expected due to the intensified electron transfer from Fe to B/P, and the glass forming ability decreases correspondingly. The idea of enhancing electron transfer can be applied to the development of other Fe-based amorphous alloys for the purpose of larger saturation magnetization.

  18. Thermo-stability of the oral rabies virus vaccines SAD B19 and SAD P5/88.

    PubMed

    Vos, A; Neubert, A

    2002-10-01

    The thermo-stability of two widely used oral rabies vaccine viruses, SAD B19 and SAD P5/88, was examined under various laboratory and field conditions. In the laboratory, both vaccine viruses were kept at 35 degrees C and titrated after 3 days. The titer of both vaccine viruses was also determined after 4 and 7 days when stored at 20 degrees C. Furthermore, vaccine baits were placed in three different micro-environments during two successive 21-day observation periods (11.9.01-2.10.01 and 2.10.01-23.10.01); (i) wooded area--location A, (ii) grassy meadow--location B, and (iii) barren soil--location C. Baits were re-collected 0, 3, 6, 9, 12, 15 and 21 days after distribution and the vaccine virus was subsequently titrated. The temperature stress for the vaccine baits was highest at location C, followed by location B and the lowest at location A. The vaccine baits were exposed to higher temperatures and higher temperature fluctuations at location B and C during trial I than during trial II. However, for both vaccines the loss of titer was more pronounced during trial II than during trial I. It is therefore suggested that under the given climatic conditions, the stability of the virus was hardly influenced by the temperatures and temperature fluctuations. PMID:12448972

  19. Genotypic Identification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In comparison with traditional, phenotype-based procedures for detection and identification of foodborne pathogen Listeria monocytogenes, molecular techniques are superior in terms of sensitivity, specificity and speed. This chapter provides a comprehensive review on the use of molecular methods for...

  20. 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. PMID:26475096

  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. 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...

  3. Genotype-specific risk factors for Staphylococcus aureus in Swiss dairy herds with an elevated yield-corrected herd somatic cell count.

    PubMed

    Berchtold, B; Bodmer, M; van den Borne, B H P; Reist, M; Graber, H U; Steiner, A; Boss, R; Wohlfender, F

    2014-01-01

    Bovine mastitis is a frequent problem in Swiss dairy herds. One of the main pathogens causing significant economic loss is Staphylococcus aureus. Various Staph. aureus genotypes with different biological properties have been described. Genotype B (GTB) of Staph. aureus was identified as the most contagious and one of the most prevalent strains in Switzerland. The aim of this study was to identify risk factors associated with the herd-level presence of Staph. aureus GTB and Staph. aureus non-GTB in Swiss dairy herds with an elevated yield-corrected herd somatic cell count (YCHSCC). One hundred dairy herds with a mean YCHSCC between 200,000 and 300,000cells/mL in 2010 were recruited and each farm was visited once during milking. A standardized protocol investigating demography, mastitis management, cow husbandry, milking system, and milking routine was completed during the visit. A bulk tank milk (BTM) sample was analyzed by real-time PCR for the presence of Staph. aureus GTB to classify the herds into 2 groups: Staph. aureus GTB-positive and Staph. aureus GTB-negative. Moreover, quarter milk samples were aseptically collected for bacteriological culture from cows with a somatic cell count ≥150,000cells/mL on the last test-day before the visit. The culture results allowed us to allocate the Staph. aureus GTB-negative farms to Staph. aureus non-GTB and Staph. aureus-free groups. Multivariable multinomial logistic regression models were built to identify risk factors associated with the herd-level presence of Staph. aureus GTB and Staph. aureus non-GTB. The prevalence of Staph. aureus GTB herds was 16% (n=16), whereas that of Staph. aureus non-GTB herds was 38% (n=38). Herds that sent lactating cows to seasonal communal pastures had significantly higher odds of being infected with Staph. aureus GTB (odds ratio: 10.2, 95% CI: 1.9-56.6), compared with herds without communal pasturing. Herds that purchased heifers had significantly higher odds of being infected with

  4. 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

  5. Hepatitis C virus genotypes in Myanmar.

    PubMed

    Win, Nan Nwe; Kanda, Tatsuo; Nakamoto, Shingo; Yokosuka, Osamu; Shirasawa, Hiroshi

    2016-07-21

    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

  6. 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

  7. Assessment of the F9 genotype-specific FIX inhibitor risks and characterization of 10 novel severe F9 defects in the first molecular series of Argentine patients with haemophilia B

    PubMed Central

    Radic, Claudia Pamela; Rossetti, Liliana Carmen; Abelleyro, Miguel Martín; Candela, Miguel; Bianco, Raúl Pérez; Pinto, Miguel de Tezanos; Larripa, Irene Beatriz; Goodeve, Anne; De Brasi, Carlos Daniel

    2014-01-01

    In Haemophilia B (HB) (factor IX (FIX) deficiency), F9 genotype largely determines clinical phenotype. Aimed to characterise Argentine families with HB, this study presents F9 genotype frequencies and their specific FIX inhibitor risk and 10 novel F9 mutations. Ninety-one DNA samples from HB patients and relatives were subjected to a new scheme: a primary screen for large deletions, a secondary screen for point mutations using conformation sensitive gel electrophoresis, DNA-sequencing and bioinformatic analysis. Our unbiased HB population (N=52)(77% with severe, 11.5% moderate and 11.5% mild HB) showed 32 missense (61.5%) including three novel mutations predicting specific structural/functional defects in silico, 7 nonsense (13.5%)(one novel), 5 large deletions, 4 splice including three novel mutations affecting predicted splicing scores, 3 indels (two novel) and one Leiden mutation. Our comprehensive HB population included five patients with long-lasting FIX inhibitors: three nonsense (p.E35* (novel), p.R75*, p.W240*) and two entire-F9 deletions. A further patient with an indel (p.A26Rfs*14) developed transient inhibitors. A case-control analysis, based on our global prevalence of 3.05% for developing inhibitors in HB revealed that missense mutations were associated with a low risk odds ratio (OR) of 0.05 and a prevalence of 0.39%, whereas nonsense and entire-F9 deletions had significantly higher risks (OR 11.0 and 32.7) and prevalence (14.3% and 44.5%, respectively). Our cost-effective practical approach enabled identification of the causative mutation in all 55 Argentine families with HB, analysis of the molecular pathology of novel F9 defects and determination of mutation-associated FIX inhibitor risks. PMID:23093250

  8. Increased expression and secretion of interleukin-6 in human parvovirus B19 non-structural protein (NS1) transfected COS-7 epithelial cells.

    PubMed

    Hsu, T-C; Tzang, B-S; Huang, C-N; Lee, Y-J; Liu, G-Y; Chen, M-C; Tsay, G J

    2006-04-01

    Human parvovirus B19 (B19) has been associated with a variety of autoimmune diseases, including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We have demonstrated previously that B19 non-structural protein (NS1) induced apoptosis through the mitochondria cell death pathway in COS-7 epithelial cells and that B19 NS1 may play a role in the pathogenesis of autoimmune diseases. In order to examine the expression profiles of cytokines and chemokines in B19 NS1 transfected COS-7 cells, we constructed the NS1 gene in the pEGFP-C1 vector named enhanced green fluorescence protein gene (EGFP)-NS1. COS-7 cells were transfected with EGFP or EGFP-NS1 plasmid. The expression profiles of cytokines and chemokines, including interleukin (IL)-1beta, IL-5, IL-6, IL-8, IL-10, tumour necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, granulocyte-macrophage colony-stimulating factor (GM-CSF), growth-related oncogene alpha (GROalpha), interferon gamma-inducible protein (IP)-10, stromal cell derived factor (SDF)-1, macrophage inflammatory protein (MIP)-1beta, monocyte chemoattractant protein (MCP)-1, regulated upon activation normal T cell expressed and secreted (RANTES), Fractalkine, CX3CR1, CCR2, CCR5 and CCR11 were examined in COS-7 cells, EGFP and EGFP-NS1 transfected cells using enzyme-linked immunosorbent assay (ELISA) or reverse transcription-polymerase chain reaction (RT-PCR). Increased expression and levels of IL-6 were found in EGFP-NS1 transfected cells using RT-PCR and ELISA. There were no significant increases in the expression of IL-1beta, IL-8, IP-10, SDF-1, RANTES, Fractalkine, CX3CR-1, CCR2, CCR5, CCR11, TNF-alpha, GM-CSF and TGF-beta using RT-PCR. There were no significantly increased levels of IL-5, IL-10, TNF-alpha, TGF-beta, GROalpha, MIP-1beta and MCP-1 found by ELISA in this study. Our results show that increased expression and secretion of IL-6 in B19 NS1 transfected epithelial cells may play a role in the pathogenesis of

  9. Genotypic differences in behavioural entropy: unpredictable genotypes are composed of unpredictable individuals

    PubMed Central

    Stamps, Judy A.; Saltz, Julia B.; Krishnan, V.V.

    2013-01-01

    Intra-genotypic variability (IGV) occurs when individuals with the same genotype, raised in the same environment and then tested under the same conditions, express different trait values. Game theoretical and bet-hedging models have suggested two ways that a single genotype might generate variable behaviour when behavioural variation is discrete rather than continuous: behavioural polyphenism (a genotype produces different types of individuals, each of which consistently expresses a different type of behaviour) or stochastic variability (a genotype produces one type of individual who randomly expresses different types of behaviour over time). We first demonstrated significant differences across 14 natural genotypes of male Drosophila melanogaster in the variability (as measured by entropy) of their microhabitat choice, in an experiment in which each fly was allowed free access to four different types of habitat. We then tested four hypotheses about ways that within-individual variability might contribute to differences across genotypes in the variability of microhabitat choice. There was no empirical support for three hypotheses (behavioural polymorphism, consistent choice, or time-based choice), nor could our results be attributed to genotypic differences in activity levels. The stochastic variability hypothesis accurately predicted the slope and the intercept of the relationship across genotypes between entropy at the individual level and entropy at the genotype level. However, our initial version of the stochastic model slightly but significantly overestimated the values of individual entropy for each genotype, pointing to specific assumptions of this model that might need to be adjusted in future studies of the IGV of microhabitat choice. This is among a handful of recent studies to document genotypic differences in behavioural IGV, and the first to explore ways that genotypic differences in within-individual variability might contribute to differences among

  10. 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.

  11. Nonstructural protein (NS1) of human parvovirus B19 stimulates host innate immunity and blunts the exogenous type I interferon signaling in vitro.

    PubMed

    Wu, Jianqin; Chen, Xu; Ye, Haiyan; Yao, Min; Li, Shilin; Chen, Limin

    2016-08-15

    B19 virus is a non-enveloped DNA virus and belongs to the family of parvoviridae. There are two large open reading frames (ORFs), nonstructural protein (NS1) and two capsid proteins (VP1 and VP2). Host innate immune responses form the first line of defense against many pathogen invasion. How B19 virus, especially its encoded viral proteins interacts with host innate immune system remains unknown. In this study we aim to investigate the effect of NS1 on the host innate immune response and exogenous type I IFN signaling. Here we found that the type I IFN can be stimulated by NS1. Interestingly, NS1 also plays an important role in inhibiting the exogenous type I IFN signaling at p-STAT1, ISRE and ISGs levels. We concluded that NS1 may play pivotal role in evading the host immune surveillance. Our data shed novel light on the pathogenesis of B19 viral infection and virus evasion strategies. PMID:27270128

  12. Simultaneous Genotyping and Quantification of Hepatitis B Virus for Genotypes B and C by Real-Time PCR Assay▿

    PubMed Central

    Zhao, Yao; Zhang, Xiu-Yu; Guo, Jin-Jun; Zeng, Ai-Zhong; Hu, Jie-Li; Huang, Wen-Xiang; Shan, You-Lan; Huang, Ai-Long

    2010-01-01

    Hepatitis B virus (HBV) is an important cause of human chronic liver diseases and is a major public health problem. Viral load and HBV genotype play critical roles in determining clinical outcomes and response to antiviral treatment in hepatitis B patients. Viral genotype detection and quantification assays are currently in use with different levels of effectiveness. In this study, the performance of a real-time genotyping and quantitative PCR (GQ-PCR)-based assay was evaluated. Through the use of genotype-specific primers and probes, this assay provides simultaneous identification and quantification of genotypes B and C in a single reaction. Our GQ-PCR correctly identified all predefined genotypes B and C, and no cross-reaction between genotypes B and C were observed. The GQ-PCR identified more cases of HBV infections with mixed genotypes B and C than direct sequencing did. Samples from 127 HBV-infected Chinese patients were genotyped with GQ-PCR, revealing 56.7% HBV as genotype B, 13.4% as genotype C, and 29.8% as mixed genotypes B and C. This assay provides a reliable, efficient, and cost-effective means for quantification of the B and C genotypes of HBV in single or mixed infections. This assay is suitable for sequential monitoring of viral load levels and for determining the relationship between the genotype viral load and stage of disease in Asians. PMID:20720032

  13. Smarter clustering methods for SNP genotype calling

    PubMed Central

    Lin, Yan; Tseng, George C.; Cheong, Soo Yeon; Bean, Lora J. H.; Sherman, Stephanie L.; Feingold, Eleanor

    2008-01-01

    Motivation: Most genotyping technologies for single nucleotide polymorphism (SNP) markers use standard clustering methods to ‘call’ the SNP genotypes. These methods are not always optimal in distinguishing the genotype clusters of a SNP because they do not take advantage of specific features of the genotype calling problem. In particular, when family data are available, pedigree information is ignored. Furthermore, prior information about the distribution of the measurements for each cluster can be used to choose an appropriate model-based clustering method and can significantly improve the genotype calls. One special genotyping problem that has never been discussed in the literature is that of genotyping of trisomic individuals, such as individuals with Down syndrome. Calling trisomic genotypes is a more complicated problem, and the addition of external information becomes very important. Results: In this article, we discuss the impact of incorporating external information into clustering algorithms to call the genotypes for both disomic and trisomic data. We also propose two new methods to call genotypes using family data. One is a modification of the K-means method and uses the pedigree information by updating all members of a family together. The other is a likelihood-based method that combines the Gaussian or beta-mixture model with pedigree information. We compare the performance of these two methods and some other existing methods using simulation studies. We also compare the performance of these methods on a real dataset generated by the Illumina platform (www.illumina.com). Availability: The R code for the family-based genotype calling methods (SNPCaller) is available to be downloaded from the following website: http://watson.hgen.pitt.edu/register. Contact: liny@upmc.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:18826959

  14. 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

  15. 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

  16. 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. PMID:26035838

  17. Molecular characterization of antigen B2 subunit in two genotypes of Echinococcus granulosus from Indian bubaline isolates, its stage specific expression and serological evaluation.

    PubMed

    Pan, D; Bera, A K; Bandyopadhyay, S; Das, S; Rana, T; Das, S K; Bandyopadhyay, S; Manna, B; Bhattacharya, D

    2011-03-01

    Echinococcus granulosus is a parasitic helminth which affects both man and animals. During infection with larval stage of the organism secretory and membrane-bound (S/M) proteins play a meaningful role for evasion of immune system. Antigen B (AgB) is one of them. Present investigation has defined sequence diversity of AgB2 subunit of cattle and buffalo isolates of the organism. A total of 55 isolates were screened by polymerase chain reaction based single stranded conformation polymorphism (PCR-SSCP). Subsequently, six conformers could be detected. Based on predicted amino acid sequences of 90 amino acid residues, three clusters could be deduced. Sequence information of two buffalo isolates was homologous to AgB4 indicating gene switching phenomenon in between closely related isoforms. Numerical value of Tajima's D test proved negative selection pressure. Using artificial neural network (ANN), B cell linear epitope and stretches of agretope were predicted. Three clusters could be defined on the basis of B cell linear epitope. Out of three clusters, two showed more than 50% binding propensity with same MHCII alleles whereas, cluster 3 exhibited binding propensity with other MHCII alleles (DRB1_1501, DRB1_1502). Relative expression of AgB2 was more in active cysts (1.636 ± 0.092) followed by degenerating (0.449 ± 0.037) and calcified (0.255 ± 0.008). This result suggested that relative expression of AgB2 declines with progression of the disease. Using recombinant AgB2 sensitivity, specificity and accuracy of the ELISA test was 96.7, 94.7 and 95.9%, respectively. No cross reactivity was found with common cestode and trematode infected cattle and buffalo because cross reactive antigen was expressed intracellularly. Finally, this was concluded that AgB2 is the suitable immunological marker for detection, diagnosis and progression of the disease. PMID:20852940

  18. Transmission and persistence of Ceratonova shasta genotypes in Chinook salmon.

    PubMed

    Hurst, Charlene N; Wong, Peter; Hallett, Sascha L; Ray, R Adam; Bartholomew, Jerri L

    2014-12-01

    Ceratonova shasta is a myxozoan parasite of salmon and trout transmitted by waterborne actinospores. Based on DNA sequence data and host specificity, 4 distinct parasite genotypes are recognized. Genotypes I and II are common in the lower reaches of the Klamath River, Oregon-California, but only infection by genotype I causes mortality in Chinook salmon. We conducted sentinel fish exposures and determined genotype composition in river water during exposure, and in fish gills, intestine, and tank water post-exposure to determine whether: (1) transmission of parasites having different genotypes is host-specific and (2) all transmitted genotypes persist in the host through to release as waterborne stages. Initial parasite transmission to the fish host appears indiscriminant, since we detected both genotypes I and II in 83.6% of the fish gills sampled. However, only genotype I was detected in fish that succumbed to infection, while both genotypes persisted in fish that survived. Persistence was likely dependent on exposure dose, initial infection type (mixed or single) and infection outcome (mortality or survival). The transmission of both genotypes to a majority of Chinook salmon and the persistence of multiple genotypes raises questions about how infection with mixed genotypes could result in within-host interactions that affect disease severity. PMID:24945751

  19. 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

  20. 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

  1. Human parvovirus B19 non-structural protein (NS1) induces apoptosis through mitochondria cell death pathway in COS-7 cells.

    PubMed

    Hsu, Tsai-Ching; Wu, Wen-Jun; Chen, Meng-Chi; Tsay, Gregory J

    2004-01-01

    Human parvovirus B19 has been found in various tissues in addition to erythroid lineage cells, and non-structural protein (NS1) is reported to induce cytotoxicity and apoptosis in erythroid lineage cells, but the mechanism in non-permissive cells is still unclear. To address this issue, we have constructed the NS1 gene in a cytomegalovirus episomal vector, pEGFP-C1 and transfected it into monkey epithelial cells, COS-7. EGFP-NS1 expression in transfected cells was monitored and assessed by fluorescence microscopy, RT-PCR and Western blot. The flow cytometric analysis showed that the NS1-transfected cells were arrested at G1 phase by paclitaxel treatment and there was increased apoptosis. The expression of p53, an important molecule in apoptosis and cell cycle regulation, and its downstream cell cycle kinase inhibitors p16(INK4) and p21(WAF1/CIP1) were up-regulated in the NS1-transfected cells. Also, increased expression of the pro-apoptotic Bcl-2 members Bax, Bad and activation of caspase 3 and caspase 9, but not the activation of caspase 8 or Fas were detected in the NS1-transfected cells. p53-induced Bax expression and subsequent activation of caspase 9 is probably the apoptotic pathway in NS1-transfected cells since activation of the caspase 9 was suppressed by the p53 inhibitor and apoptosis was significantly inhibited by the caspase 9 inhibitor. Our results suggest that the cell death of the NS1-transfected cells is associated with mitochondria related apoptosis. These findings might provide alternative information for further study and characterization of B19 NS1 protein in B19 non-permissive cells. PMID:15370668

  2. Human platelet antigen 1 (HPA 1) genotyping with 5' nuclease assay and sequence-specific primers reveals a single nucleotide deletion in intron 2 of the HPA 1a allele of platelet glycoprotein IIIa.

    PubMed

    Kjaer, Killie Mette; Jaegtvik, Sissel; Husebekk, Anne; Skogen, Bjorn

    2002-05-01

    We have established a 5' nuclease assay (5' NA) for human platelet antigen (HPA) 1a/b allelic discrimination. The assay is based on the simultaneous amplification and detection of the two targets in a one-tube system. The results are read optically, immediately after termination of the polymerase chain reaction (PCR), and no post-PCR processing is necessary. This genotyping procedure is less time-consuming and cheaper than our conventional sequence-specific primer PCR (SSP-PCR), which is run as a two-tube test, with verification of the results after electrophoresis in agarose gel. The reduction of analytical steps, simplification of the procedure and potential for automation were important advantages for our choice of system. This test system is more suitable for large-scale testing and fits better for our screening programme for HPA 1bb determination. DNA from 1093 individuals were tested in parallel with the SSP-PCR and the 5' NA. One thousand and ninety-one samples gave identical results in SSP-PCR and 5' NA. Upon repeated testing, two samples consistently came out as HPA 1bb in SSP-PCR and HPA 1ab in 5' NA. DNA sequencing revealed a defect located in an intronic area that corresponds to the consensus primer used for the SSP-PCR HPA 1a typing. PMID:11972525

  3. Strong HCV NS3/4a, NS4b, NS5a, NS5b-specific cellular immune responses induced in Rhesus macaques by a novel HCV genotype 1a/1b consensus DNA vaccine

    PubMed Central

    Latimer, Brian; Toporovski, Roberta; Yan, Jian; Pankhong, Panyupa; Morrow, Matthew P; Khan, Amir S; Sardesai, Niranjan Y; Welles, Seth L; Jacobson, Jeffrey M; Weiner, David B; Kutzler, Michele A

    2014-01-01

    Chronic HCV is a surreptitious disease currently affecting approximately 3% of the world's population that can lead to liver failure and cancer decades following initial infection. However, there are currently no vaccines available for the prevention of chronic HCV. From patients who acutely resolve HCV infection, it is apparent that a strong and broad cytotoxic T lymphocyte (CTL) response is important in HCV clearance. DNA vaccines are naked plasmid DNA molecules that encode pathogen antigens to induce a pathogen-specific immune response. They are inexpensive to produce and have an excellent safety profile in animals and humans. Additionally, DNA vaccines are able to induce strong CTL responses, making them well-suited for an HCV vaccine. We aimed to maximize vaccine recipients' opportunity to induce a broad T cell response with a novel antigenic sequence, multi-antigen vaccine strategy. We have generated DNA plasmids encoding consensus sequences of HCV genotypes 1a and 1b non-structural proteins NS3/4a, NS4b, NS5a, and NS5b. Rhesus macaques were used to study the immunogenicity of these constructs. Four animals were immunized 3 times, 6 weeks apart, at a dose of 1.0mg per antigen construct, as an intramuscular injection followed by in vivo electroporation, which greatly increases DNA uptake by local cells. Immune responses were measured 2 weeks post-immunization regimen (PIR) in immunized rhesus macaques and showed a broad response to multiple HCV nonstructural antigens, with up to 4680 spot-forming units per million peripheral blood mononuclear cells (PBMCs) as measured by Interferon-γ ELISpot. In addition, multiparametric flow cytometry detected HCV-specific CD4+ and CD8+ T cell responses by intracellular cytokine staining and detected HCV-specific CD107a+/GrzB+ CD8+ T cells indicating an antigen specific cytolytic response 2 weeks PIR compared with baseline measurements. At the final study time point, 6 weeks PIR, HCV-specific CD45RA- memory-like T cells

  4. 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

  5. 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

  6. 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

    2016-01-01

    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

  7. 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 Central

    Bagaglio, Sabrina; Andolina, Andrea; Merli, Marco; Uberti-Foppa, Caterina; Morsica, Giulia

    2016-01-01

    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

  8. Association of Transferable Quinolone Resistance Determinant qnrB19 with Extended-Spectrum β-Lactamases in Salmonella Give and Salmonella Heidelberg in Venezuela

    PubMed Central

    González, Fanny

    2013-01-01

    Four nontyphoidal Salmonella strains with resistance to extended-spectrum cephalosporins and nonclassical quinolone resistance phenotype were studied. Two S. Give were isolated from pediatric patients with acute gastroenteritis, and two S. Heidelberg were recovered from raw chicken meat. Phenotypic characterization included antimicrobial susceptibility testing and detection of extended-spectrum β-lactamases (ESBLs) by the double-disc synergy method. The detection of quinolone resistance-determining regions (QRDR) of gyrA, gyrB, and gyrC genes, blaESBLs genes, and plasmid-mediated quinolone resistance (PMQR) determinants was carried out by molecular methods. Plasmid analysis included Southern blot and restriction patterns. Transferability of resistance genes was examined by transformation. blaTEM-1 + blaSHV-12 genes were detected in S. Give SG9611 and blaTEM-1 + blaCTX-M-2 in the other three strains: S. Give SG9811, S. Heidelberg SH7511, and SH7911. Regardless of origin and serovars, the qnrB19 gene was detected in the 4 strains studied. All determinants of resistance were localized in plasmids and successfully transferred by transformation. This study highlights the circulation of qnrB19 associated with blaTEM-1, blaSHV-12, and blaCTX-M-2 in S. Give and S. Heidelberg in Venezuela. The recognition of factors associated with increasing resistance and the study of the molecular mechanisms involved can lead to a more focused use of antimicrobial agents. PMID:24187555

  9. 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.

  10. Gianotti-Crosti syndrome associated with Ebstein-Barr virus and Parvovirus B-19 coinfection in a male adult: case report and review of the literature.

    PubMed

    Stojkovic-Filipovic, Jelena; Skiljevic, Dusan; Brasanac, Dimitrije; Medenica, Ljiljana

    2016-02-01

    Gianotti-Crosti syndrome (GCS) is a self-limiting, mostly childhood-appearing, cutaneous eruption with characteristic symmetric areal distribution. The original cases, described by Gianotti in 1955, were associated with hepatitis B virus infection, but other viral and bacterial infections, as well as immunizations, have been implied in etiology of this condition. Adult cases are rare and have been reported almost exclusively in women. We present the case of a 20-year-old Caucasian man who had typical clinical presentation: monomorphic pale, pink-to-flesh - colored or erythematous papules and papulovesicles localized symmetrically over the extensor surfaces of the extremities, buttocks and the face; some lesions were detected on knees, elbows and palms, as well. Laboratory tests revealed slight bilirubin and alanine aminotransaminase elevation. Serology tests demonstrated antibodies against Epstein-Barr virus and parvovirus B-19. Histology of skin biopsy specimens revealed a vesicular dermatitis with perivascular lymphocytic infiltrate. Oral and topical corticosteroids and oral antihistamines led to complete resolution of lesions in 3 weeks. GCS is rare in adults, especially men. To the best of our knowledge, this is the fifth male adult case and the first with Parvovirus B-19 and EBV coinfection. PMID:25034095

  11. The adenovirus E1A proteins induce apoptosis, which is inhibited by the E1B 19-kDa and Bcl-2 proteins.

    PubMed Central

    Rao, L; Debbas, M; Sabbatini, P; Hockenbery, D; Korsmeyer, S; White, E

    1992-01-01

    Cooperation between the adenovirus E1A and E1B oncogenes is required for transformation of primary quiescent rodent cells. Although expression of E1A alone will stimulate cell proliferation sufficient to initiate transformed focus formation, proliferation fails to be sustained and foci degenerate. Coexpression of either the 19-kDa or 55-kDa E1B oncoproteins with E1A permits high-frequency transformation by overcoming this cytotoxic response. Without E1B 19-kDa protein expression, however, transformants remain susceptible to induction of cell death. Rapid loss of viability is coincident with nucleolytic cleavage of DNA in intranucleosomal regions and chromatin condensation, hallmarks of programmed cell death (apoptosis). Furthermore, overexpression of a known suppressor of apoptosis, the Bcl-2 protooncogene, can rescue E1A-induced focus degeneration. Thus E1A-dependent stimulation of cell proliferation is accompanied by apoptosis and thereby insufficient to singly induce transformation. High-frequency transformation requires a second function encoded by the E1B 19-kDa protein to block apoptosis. Images PMID:1457005

  12. 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: ...

  13. HCV genotypes in Morocco.

    PubMed

    Benani, A; El-Turk, J; Benjelloun, S; Sekkat, S; Nadifi, S; Hda, N; Benslimane, A

    1997-08-01

    To determine the hepatitis C virus (HCV) genotypes circulating in Morocco, virus isolates from 105 chronically infected and 19 hemodialysis patients were examined using the line probe assay. Genotypes 1 and 2 only were found among Moroccan patients. Subtypes 1b (47.6%) and 2a/2c (37.1%) were the most common, whereas subtype 1a (2.8%) was less common. Among the hemodialysis patients, only genotype 1 was found with a prevalence of 68.4% for subtype 1b and 15.8% for the subtype 1a. It was also shown that the HCV genotypes distribution varies with age in both studied populations. Subtype 1b was most prevalent among older patients, whereas subtype 2a/2c was mainly found among younger ones. Although Morocco belongs to the African continent, the circulating HCV strains are similar to those observed in some American and European countries. PMID:9260687

  14. 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 ...

  15. 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

  16. X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. X. Heterozygous effects of multilocus deletion mutations of genotype ad-3A or ad-3B.

    PubMed

    de Serres, F J; Overton, L K; Sadler, B M

    1992-05-01

    Previous studies on X-ray-induced irreparable adenine-3 mutations (designated [ad-3]IR), induced in heterokaryon 12 of Neurospora crassa, demonstrated that they were not recessive and exhibited heterozygous effects in terms of markedly reduced linear growth rates (de Serres, 1965). Complementation tests with a series of tester strains carrying multilocus deletion mutations in the ad-3 and immediately adjacent genetic regions demonstrated that X-ray-induced irreparable mutations map, in the main part, as a series of overlapping multilocus deletion mutations that extend both proximally and distally into the immediately adjacent genetic regions, as well as into the 'X' region (a region of unknown, but essential function) between ad-3A and ad-3B (de Serres, 1968, 1989). Further studies (de Serres and Miller, 1988) have shown that the heterozygous effects of multilocus deletion mutations in the ad-3 region can be modified genetically and biochemically. In the present paper, the heterozygous effects of X-ray-induced multilocus deletion mutations of genotype ad-3A or ad-3B, induced in heterokaryon 12 (Webber and de Serres, 1965; de Serres, 1988, 1989), have been determined. These data show that 57.7% (15/26) of X-ray-induced multilocus deletion mutations covering the ad-3A locus have heterozygous effects, in terms of reduced linear growth rates, in forced dikaryons with a gene/point mutant at the ad-3B locus and 80.0% (20/25) in forced dikaryons with a multilocus deletion mutation covering the ad-3B locus. In addition, 35.1% (20/57) of X-ray-induced multilocus deletion mutations covering the ad-3B locus have heterozygous effects in forced dikaryons with a gene/point mutant at the ad-3A locus, and 100.0% (35/35) in forced dikaryons with a multilocus deletion mutation covering the ad-3A locus. These results demonstrate that the dominant or recessive characteristics of X-ray-induced specific-locus mutations resulting from multilocus deletion mutations are allele specific

  17. Development of an improved genotyping assay for the detection of hepatitis C virus genotypes and subtypes in Pakistan.

    PubMed

    Idrees, Muhammad

    2008-06-01

    A new genotyping system was established for the specific detection of HCV genotypes 1a, 1b, 1c, 2a, 2b, 2c, 3a, 3b, 3c, 4a-h, 5a and 6a during the course of this study. The system is based on entire core region and a part of 5' noncoding region (5'NCR) with genotype-specific primers. Genotype-specific primers were designed on the basis of 114 HCV isolates. Serum samples with known genotypes were used as positive controls to validate the assay developed and to generate PCR band patterns. Band patterns generated from the clinical serum samples from HCV patients were compared to the patterns produced from these control samples. In addition, the type-specific bands were sequenced from the test patients and control clinical samples to validate further the test results. To determine sensitivity and specificity of the assay, a total 260 samples were analyzed simultaneously by this HCV genotyping method and that developed by Ohno and Murex HCV Serotyping 1-6 Assay. The system showed 79.2% concordance with Ohno's system and 65.38% with serotyping system. Samples with discordant results were sequenced and their genotypes were determined by molecular evolutionary analysis. The data indicate that the method described in this study may offer better sensitivity and specificity for the detection directly of HCV genotypes present at low levels in HCV patient samples. PMID:18423633

  18. Genotype imputation in the domestic dog.

    PubMed

    Friedenberg, S G; Meurs, K M

    2016-10-01

    Application of imputation methods to accurately predict a dense array of SNP genotypes in the dog could provide an important supplement to current analyses of array-based genotyping data. Here, we developed a reference panel of 4,885,283 SNPs in 83 dogs across 15 breeds using whole genome sequencing. We used this panel to predict the genotypes of 268 dogs across three breeds with 84,193 SNP array-derived genotypes as inputs. We then (1) performed breed clustering of the actual and imputed data; (2) evaluated several reference panel breed combinations to determine an optimal reference panel composition; and (3) compared the accuracy of two commonly used software algorithms (Beagle and IMPUTE2). Breed clustering was well preserved in the imputation process across eigenvalues representing 75 % of the variation in the imputed data. Using Beagle with a target panel from a single breed, genotype concordance was highest using a multi-breed reference panel (92.4 %) compared to a breed-specific reference panel (87.0 %) or a reference panel containing no breeds overlapping with the target panel (74.9 %). This finding was confirmed using target panels derived from two other breeds. Additionally, using the multi-breed reference panel, genotype concordance was slightly higher with IMPUTE2 (94.1 %) compared to Beagle; Pearson correlation coefficients were slightly higher for both software packages (0.946 for Beagle, 0.961 for IMPUTE2). Our findings demonstrate that genotype imputation from SNP array-derived data to whole genome-level genotypes is both feasible and accurate in the dog with appropriate breed overlap between the target and reference panels. PMID:27129452

  19. Delay of vaccinia virus-induced apoptosis in nonpermissive Chinese hamster ovary cells by the cowpox virus CHOhr and adenovirus E1B 19K genes.

    PubMed Central

    Ink, B S; Gilbert, C S; Evan, G I

    1995-01-01

    The infection of vaccinia virus in Chinese hamster ovary (CHO) cells produces a rapid shutdown in protein synthesis, and the infection is abortive (R.R. Drillien, D. Spehner, and A. Kirn, Virology 111:488-499, 1978; D.E. Hruby, D.L. Lynn, R. Condit, and J.R. Kates, J. Gen. Virol. 47:485-488, 1980). Cowpox virus, which can productively infect CHO cells, had previously been shown to contain a host range gene, CHOhr, which confers on vaccinia virus the ability to replicate in CHO cells (D. Spehner, S. Gillard, R. Drillien, and A. Kirn, J. Virol. 62:1297-1304, 1988). We found that CHO cells underwent apoptosis when infected with vaccinia virus. The expression of the CHOhr gene in vaccinia virus allowed for the expression of late virus genes. CHOhr also delayed or prevented vaccinia virus-induced apoptosis in CHO cells such that there was sufficient time for replication of the virus before the cell died. The E1B 19K gene from adenovirus also delayed vaccinia virus-induced apoptosis; however, there was no detectable expression of late virus genes. Furthermore, E1B 19K also delayed cell death in CHO cells which had been productively infected with vaccinia virus. This study identifies a new antiapoptotic gene from cowpox virus, CHOhr, for which the protein contains an ankyrin-like repeat and shows no significant homology to other proteins. This work also indicates that an antiapoptotic gene from one virus family can delay cell death in an infection of a virus from a different family. PMID:7815529

  20. 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...

  1. Blood Group ABO Genotyping in Paternity Testing

    PubMed Central

    Bugert, Peter; Rink, Gabriele; Kemp, Katharina; Klüter, Harald

    2012-01-01

    Background The ABO blood groups result from DNA sequence variations, predominantly single nucleotide and insertion/deletion polymorphisms (SNPs and indels), in the ABO gene encoding a glycosyltransferase. The ABO blood groups A1, A2, B and O predominantly result from the wild type allele A1 and the major gene variants that are characterized by four diallelic markers (261G>del, 802G>A, 803G>C, 1061C>del). Here, we were interested to evaluate the impact of ABO genotyping compared to ABO phenotyping in paternity testing. Methods The major ABO alleles were determined by PCR amplification with sequence-specific primers (PCR-SSP) in a representative sample of 1,335 blood donors. The genotypes were compared to the ABO blood groups registered in the blood donor files. Then, the ABO phenotypes and genotypes were determined in 95 paternity trio cases that have been investigated by 12 short tandem repeat (STR) markers before. We compared statistical parameters (PL, paternity likelihood; PE, power of exclusion) of both blood grouping approaches. Results The prevalence of the major ABO alleles and genotypes corresponded to the expected occurrence of ABO blood groups in a Caucasian population. The low resolution genotyping of 4 diallelic markers revealed a correct genotype-phenotype correlation in 1,331 of 1,335 samples (99.7%). In 60 paternity trios with confirmed paternity of the alleged father based on STR analysis both PL and PE of the ABO genotype was significantly higher than of the ABO phenotype. In 12 of 35 exclusion cases (34.3%) the ABO genotype also excluded the alleged father, whereas the ABO phenotype excluded the alleged father only in 7 cases (20%). Conclusion In paternity testing ABO genotyping is superior to ABO phenotyping with regard to PL and PE, however, ABO genotyping is not sufficient for valid paternity testing. Due to the much lower mutation rate compared to STR markers, blood group SNPs in addition to anonymous SNPs could be considered for future

  2. 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 ...

  3. 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

  4. 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

  5. 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

  6. Mass genotyping by sequencing technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Large scale genotyping of a moderate number of loci is cost prohibitive with current chip-based technologies. We demonstrate the ability to use next generation sequencing technologies to genotype many DNA samples for a moderate number of loci – a mass genotyping by sequencing technology (MGST). Ou...

  7. 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

  8. 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

  9. 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

  10. miR-103 Regulates Oxidative Stress by Targeting the BCL2/Adenovirus E1B 19 kDa Interacting Protein 3 in HUVECs

    PubMed Central

    Xu, Mao-Chun; Gao, Xiu-Fang; Ruan, Changwu; Ge, Zhi-Ru; Lu, Ji-De; Zhang, Jian-Jun; Zhang, Yu; Wang, Lu; Shi, Hai-Ming

    2015-01-01

    Oxidative stress plays a critical role in cardiovascular diseases. Salidroside, a glycoside from Rhodiola rosea, has been used as an antioxidative therapy for oxidative injury in cardiac diseases. However, the mechanism underlying its antioxidant effect needs to be elucidated. Treatment of HUVECs with H2O2 significantly decreased the expression of miR-103 in a dose- and time-dependent manner, whereas pretreatment with salidroside significantly inhibited this decrease. Subsequent analysis showed that overexpression of miR-103 abrogated cell activity and ROS production induced by H2O2. Bcl2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) was determined to be a novel miR-103 target in HUVECs. Interestingly, H2O2 treatment upregulated BNIP3 expression; in turn, this effect was inhibited by pretreatment with salidroside. Further studies confirmed that the knockdown of BNIP3 enhanced cell activity and suppressed the ROS production induced by H2O2. These results demonstrated for the first time that salidroside protects HUVECs in part by upregulating the expression of miR-103, which mediates BNIP3 downregulation and plays an important role in the cytoprotective actions. PMID:26000071

  11. Development and applications of low-cost, high-throughput genotyping

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Most current genotyping systems are too expensive to justify routine genotyping of all animals in livestock breeding and/or production. Therefore, a low-cost genotyping method was developed in which barcodes identifying individuals are added to DNA fragments programmed by specific loci. Next gener...

  12. Distribution of hepatitis C virus genotypes in a diverse US integrated health care population.

    PubMed

    Manos, M Michele; Shvachko, Valentina A; Murphy, Rosemary C; Arduino, Jean Marie; Shire, Norah J

    2012-11-01

    Hepatitis C virus (HCV) genotypes influence response to therapy, and recently approved direct-acting antivirals are genotype-specific. Genotype distribution information can help to guide antiviral development and elucidate infection patterns. HCV genotype distributions were studied in a diverse cross-section of patients in the Northern California Kaiser Permanente health plan. Associations between genotype and race/ethnicity, age, and sex were assessed with multivariate logistic regression models. The 10,256 patients studied were median age 56 years, 62% male, 55% White non-Hispanic. Overall, 70% were genotype 1, 16% genotype 2, 12% genotype 3, 1% genotype 4, <1% genotype 5, and 1% genotype 6. Blacks (OR 4.5 [3.8-5.5]) and Asians (OR 1.2 [1.0-1.4]) were more likely to have genotype 1 than 2/3 versus non-Hispanic Whites. Women less likely had genotype 1 versus 2/3 than did men (OR 0.86 [0.78-0.94]). Versus non-Hispanic Whites, Asians (OR 0.38 [0.31-0.46]) and Blacks (OR 0.73 [0.63-0.84]) were less likely genotype1a than 1b; Hispanics (OR 1.3 [1.1-1.5]) and Native Americans (OR 1.9 [1.2-2.8]) more likely had genotype 1a than 1b. Patients age ≥65 years less likely had genotype 1a than 1b versus those age 45-64 (OR 0.34 [0.29-0.41]). The predominance of genotype 1 among all groups studied reinforces the need for new therapies targeting this genotype. Racial/ethnic variations in HCV genotype and subtype distribution must be considered in formulating new agents and novel strategies to successfully treat the diversity of hepatitis C patients. PMID:22997077

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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. PMID:25829264

  18. Multiplexed genotyping with sequence-tagged molecular inversion probes.

    PubMed

    Hardenbol, Paul; Banér, Johan; Jain, Maneesh; Nilsson, Mats; Namsaraev, Eugeni A; Karlin-Neumann, George A; Fakhrai-Rad, Hossein; Ronaghi, Mostafa; Willis, Thomas D; Landegren, Ulf; Davis, Ronald W

    2003-06-01

    We report on the development of molecular inversion probe (MIP) genotyping, an efficient technology for large-scale single nucleotide polymorphism (SNP) analysis. This technique uses MIPs to produce inverted sequences, which undergo a unimolecular rearrangement and are then amplified by PCR using common primers and analyzed using universal sequence tag DNA microarrays, resulting in highly specific genotyping. With this technology, multiplex analysis of more than 1,000 probes in a single tube can be done using standard laboratory equipment. Genotypes are generated with a high call rate (95%) and high accuracy (>99%) as determined by independent sequencing. PMID:12730666

  19. COMT genotype, gambling activity, and cognition.

    PubMed

    Grant, Jon E; Leppink, Eric W; Redden, Sarah A; Odlaug, Brian L; Chamberlain, Samuel R

    2015-09-01

    Neuropsychological studies of adults with problem gambling indicate impairments across multiple cognitive domains. Catechol-O-methyltransferase (COMT) plays a unique role in the regulation of dopamine in the prefrontal cortex, and has been implicated in the cognitive dysfunction evident in problem gambling. This study examined adults with varying levels of gambling behavior to determine whether COMT genotype was associated with differences in gambling symptoms and cognitive functioning. 260 non-treatment-seeking adults aged 18-29 years with varying degrees of gambling behavior provided saliva samples for genotyping COMT val158met (rs4680). All subjects underwent clinical evaluations and neurocognitive assessment of decision-making, working memory, and impulsivity. The Val/Val COMT genotype was associated with the largest percentage of subjects with gambling disorder (31.8%), a rate significantly different from the Val/Met (13.2%) group (p = 0.001). The Val/Val COMT group was also associated with significantly more gambling disorder diagnostic criteria being met, greater frequency of gambling behavior, and significantly worse cognitive performance on the Cambridge Gamble Task (risk adjustment and delay aversion) and the Spatial Working Memory task (total errors). This study adds to the growing literature on the role of COMT in impulsive behaviors by showing that the Val/Val genotype was associated with specific clinical and cognitive elements among young adults who gamble, in the absence of differences on demographic measures and other cognitive domains. Future work should consider using genotyping to explore whether certain polymorphisms predict subsequent development of impulsive behaviors including gambling disorder, and treatment outcomes. PMID:26028545

  20. 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

  1. 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. PMID:25476721

  2. 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.

  3. In-situ X-ray diffraction studies of the phase transformations and structural states of B2, R and B19' phases in Ti49.5Ni50.5 alloy

    NASA Astrophysics Data System (ADS)

    Ostapenko, Marina G.; Meisner, Ludmila L.; Lotkov, Aleksandr I.; Zakharova, Margarita A.; Gudimova, Ekaterina Y.

    2015-10-01

    The martensitic transformation, Debye-Waller factor, mean-square atomic displacements and the coefficient of thermal expansion on cooling of the Ti49.5Ni50.5 shape memory alloy were examined using in-situ X-ray diffraction. It was revealed B2→R (TR ≡ T = 273 ± 10 K) along with B2→B19' (Ms ≡ 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.

  4. Genotypic diversity of beet curly top virus populations in the Western United States.

    PubMed

    Stenger, D C; McMahon, C L

    1997-07-01

    ABSTRACT The genotypic diversity of beet curly top virus (BCTV) present in the western United States has been examined by the analysis of 58 field isolates and eight laboratory or nursery isolates of the virus. Full-length clones for each isolate have been characterized for genotype by restriction endonuclease mapping. The results indicate that most of the genotypes examined may be classified as variants of the CFH, Worland, or Cal/Logan strains of BCTV. Two genotypes were recovered that appear to share certain genotypic markers of both Worland and CFH strains. Genotypic variants of the CFH and Worland strains and the two genotypes sharing markers of both strains were recovered from field isolates collected during 1994 and 1995. In contrast, the Cal/Logan strain was recovered only from isolates maintained in laboratories or nurseries. Comparisons of restriction endonuclease maps of cloned BCTV genomes revealed considerable variability both within and between strains. Although a total of 43 distinct genotypes of BCTV were identified, only 36 (84%) were recovered from field isolates. Of 37 field isolates for which more than a single clone was recovered, 16 (43%) contained more than a single genotype of one strain, whereas 4 (11%) harbored mixed infections of the CFH and Worland strains. A phylogenetic analysis using 43 characters derived from restriction endonuclease mapping data supported the grouping of 41 genotypes into three taxa consistent with the three currently recognized strains of BCTV. The relationships of the two genotypes sharing genotypic markers of both the Worland and CFH strains to other BCTV genotypes was unresolved in the phylogenetic analysis. Based on the mild symptom phenotype of the isolates from which these two genotypes were recovered and the presence of Worland genotypic markers in portions of the genome containing both cis- and trans-acting elements determining replication specificity, these two genotypes were tentatively considered as

  5. Enterocytozoon bieneusi genotypes nomenclature based on the ITS sequence– a consensus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There are 80 genotypes of Enterocytozoon bieneusi reported to infect humans, domesticated and wild mammals, and birds. Many molecularly similar genotypes cluster with specific host species and appear to be host specific such as the bovine isolates BEB1, 2, 3, 4, and 6. Others have been reported fro...

  6. Echinococcus granulosus genotypes in Iran

    PubMed Central

    Sharafi, Seyedeh Maryam; Rostami-Nejad, Mohammad; Moazeni, Mohammad; Yousefi, Morteza; Saneie, Behnam; Hosseini-Safa, Ahmad

    2014-01-01

    Hydatidosis, caused by Echinococcus granulosus is one of the most important zoonotic diseases, throughout most parts of the world. Hydatidosis is endemic in Iran and responsible for approximately 1% of admission to surgical wards. There are extensive genetic variations within E. granulosus and 10 different genotypes (G1–G10) within this parasite have been reported. Identification of strains is important for improvement of control and prevention of the disease. No new review article presented the situation of Echinococcus granulosus genotypes in Iran in the recent years; therefore in this paper we reviewed the different studies regarding Echinococcus granulosus genotypes in Iran. PMID:24834298

  7. 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

  8. 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. PMID:23110438

  9. Realizing the potential of plasma genotyping in an age of genotype-directed therapies.

    PubMed

    Luke, Jason J; Oxnard, Geoffrey R; Paweletz, Cloud P; Camidge, D Ross; Heymach, John V; Solit, David B; Johnson, Bruce E

    2014-08-01

    The identification of oncogenic driver mutations in cancer has led to the rapid rise of genotype-directed treatments such as EGFR and BRAF kinase inhibitors. Standard tumor biopsy remains a cumbersome and morbid procedure for patients, leading to a growing interest in noninvasive plasma genotyping approaches. Circulating tumor cells are of interest; however, the processing of specimens is complicated and time consuming. By comparison, cell-free DNA (cfDNA) genotyping has the potential to be convenient and relatively simple to process in a short time period. Several technologies are under development for cfDNA analysis, such as allele-specific polymerase chain reaction (PCR), coamplification at Lower Denaturation temperatures (COLD) PCR, emulsion PCR, and massively parallel sequencing. Broad clinical validity will need to be established for different assays, and clinical utility will need to be evaluated within prospective trials to determine which assays will best predict the efficacy of therapy and patient outcomes. In addition, assay standardization will be critical prior to widespread use in routine clinical practice. The Cell Free DNA Working Group, under the sponsorship of Transgenomic, was convened to evaluate the molecular assays in development and provide recommendations for application and interpretation of these tests in the context of future clinical research. The consensus commentary of the Cell Free DNA Working Group for the use of cfDNA plasma genotyping assays is presented here, including future steps in the development of these technologies. PMID:25106647

  10. Correlated genotypes in friendship networks

    PubMed Central

    Fowler, James H.; Settle, Jaime E.; Christakis, Nicholas A.

    2011-01-01

    It is well known that humans tend to associate with other humans who have similar characteristics, but it is unclear whether this tendency has consequences for the distribution of genotypes in a population. Although geneticists have shown that populations tend to stratify genetically, this process results from geographic sorting or assortative mating, and it is unknown whether genotypes may be correlated as a consequence of nonreproductive associations or other processes. Here, we study six available genotypes from the National Longitudinal Study of Adolescent Health to test for genetic similarity between friends. Maps of the friendship networks show clustering of genotypes and, after we apply strict controls for population stratification, the results show that one genotype is positively correlated (homophily) and one genotype is negatively correlated (heterophily). A replication study in an independent sample from the Framingham Heart Study verifies that DRD2 exhibits significant homophily and that CYP2A6 exhibits significant heterophily. These unique results show that homophily and heterophily obtain on a genetic (indeed, an allelic) level, which has implications for the study of population genetics and social behavior. In particular, the results suggest that association tests should include friends’ genes and that theories of evolution should take into account the fact that humans might, in some sense, be metagenomic with respect to the humans around them. PMID:21245293

  11. Canine Hip Dysplasia is Predictable by Genotyping

    PubMed Central

    Guo, Gang; Zhou, Zhengkui; Wang, Yachun; Zhao, Keyan; Zhu, Lan; Lust, George; Hunter, Linda; Friedenberg, Steven; Li, Junya; Zhang, Yuan; Harris, Stephen; Jones, Paul; Sandler, Jody; Krotscheck, Ursula; Todhunter, Rory; Zhang, Zhiwu

    2011-01-01

    Summary Objective To establish a predictive method using whole genome genotyping for early intervention in canine hip dysplasia (CHD) risk management, for the prevention of the progression of secondary osteoarthritis (OA), and for selective breeding. Design Two sets of dogs (6 breeds) were genotyped with dense SNPs covering the entire canine genome. The first set contained 359 dogs upon which a predictive formula for genomic breeding value (GBV) was derived by using their estimated breeding value (EBV) of the Norberg angle (a measure of CHD) and their genotypes. To investigate how well the formula would work for an individual dog with genotype only (without using EBV or phenotype), a cross validation was performed by masking the EBV of one dog at a time. The genomic data and the EBV of the remaining dogs were used to predict the GBV for the single dog that was left out. The second set of dogs included 38 new Labrador retriever dogs, which had no pedigree relationship to the dogs in the first set. Results The cross validation showed a strong correlation (r>0.7) between the EBV and the GBV. The independent validation showed a strong correlation (r=0.5) between GBV for the Norberg angle and the observed Norberg angle (no EBV was available for the new 38 dogs). Sensitivity, specificity, positive, and negative predictive value of the genomic data were all above 70%. Conclusions Prediction of CHD from genomic data is feasible, and can be applied for risk management of CHD and early selection for genetic improvement to reduce the prevalence of CHD in breeding programs. The prediction can be implemented before maturity, at which age current radiographic screening programs are traditionally applied, and as soon as DNA is available. PMID:21215318

  12. Newcastle disease: genotypic variation and gross pathology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There are currently 18 genotypes described for class II Newcastle disease viruses and one genotype for class I. While class I viruses are globally distributed in wild birds, the class II genotypes are found more selectively in different countries. For instance the NDV isolates in genotype V are mo...

  13. Risk factors for infection with Campylobacter jejuni flaA genotypes

    PubMed Central

    UNICOMB, L. E.; O'REILLY, L. C.; KIRK, M. D.; STAFFORD, R. J.; SMITH, H. V.; BECKER, N. G.; PATEL, M. S.; GILBERT, G. L.

    2008-01-01

    SUMMARY We aimed to explore Campylobacter genotype-specific risk factors in Australia. Isolates collected prospectively from cases recruited into a case-control study were genotyped using flaA restriction fragment-length polymorphism typing (flaA genotyping). Exposure information for cases and controls was collected by telephone interview. Risk factors were examined for major flaA genotypes using logistic and multinomial regression. Five flaA genotypes accounted for 325 of 590 (55%) cases – flaA-6b (n=129), flaA-6 (n=70), flaA-10 (n=48), flaA-2 (n=43), flaA-131 (n=35). In Australia, infections due to flaA-10 and flaA-2 were found to be significantly associated with eating non-poultry meat (beef and ham, respectively) in both case-control and inter-genotype comparisons. All major genotypes apart from flaA-10 were associated with chicken consumption in the case-control comparisons. Based on several clinical criteria, infections due to flaA-2 were more severe than those due to other genotypes. Thus genotype analysis may reveal genotype-specific niches and differences in virulence and transmission routes. PMID:18205975

  14. 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

  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. Sex and PRNP genotype determination in preimplantation caprine embryos.

    PubMed

    Guignot, F; Perreau, C; Cavarroc, C; Touzé, J-L; Pougnard, J-L; Dupont, F; Beckers, J-F; Rémy, B; Babilliot, J-M; Bed'Hom, B; Lamorinière, J M; Mermillod, P; Baril, G

    2011-08-01

    The objective of this study was to test the accuracy of genotype diagnosis after whole amplification of DNA extracted from biopsies obtained by trimming goat embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer. Whole genome amplification (WGA) was performed using Multiple Displacement Amplification (MDA). Sex and prion protein (PRNP) genotypes were determined. Sex diagnosis was carried out by PCR amplification of ZFX/ZFY and Y chromosome-specific sequences. Prion protein genotype determination was performed on codons 142, 154, 211, 222 and 240. Embryos were collected at day 7 after oestrus and biopsied either immediately after collection (blastocysts and expanded blastocysts) or after 24 h of in vitro culture (compacted morulae). Biopsied embryos were frozen by vitrification. Vitrified whole embryos were kept as control. DNA of biopsies was extracted and amplified using MDA. Sex diagnosis was efficient for 97.4% of biopsies and PRNP genotyping was determined in 78.7% of biopsies. After embryo transfer, no significant difference was observed in kidding rate between biopsied and vitrified control embryos, whereas embryo survival rate was different between biopsied and whole vitrified embryos (p = 0.032). At birth, 100% of diagnosed sex and 98.2% of predetermined codons were correct. Offspring PRNP profiles were in agreement with parental genotype. Whole genome amplification with MDA kit coupled with sex diagnosis and PRNP genotype predetermination are very accurate techniques to genotype goat embryos before transfer. These novel results allow us to plan selection of scrapie-resistant genotypes and kid sex before transfer of cryopreserved embryo. PMID:21121967

  17. [Determination of hepatitis C virus genotypes among hepatitis C patients in Eastern Black Sea Region, Turkey].

    PubMed

    Buruk, Celal Kurtuluş; Bayramoğlu, Gülçin; Reis, Ahu; Kaklıkkaya, Neşe; Tosun, Ilknur; Aydın, Faruk

    2013-10-01

    Hepatitis C virus (HCV), the major cause of transfusion-associated hepatitis, is an important public health problem in the world as well as in Turkey. HCV is grouped as six distinct genotypes and a large number of closely-related subtypes. Genotyping of HCV is an important tool for providing epidemiological data, prediction of prognosis, and optimization of antiviral therapy. This study was carried out to determine the distribution of HCV genotypes in hepatitis C patients residing in different provinces of the Eastern Black Sea Region, Turkey. A total of 304 HCV-RNA positive cases (151 male, 153 female; age range: 11-93 years, mean age: 55.2 ± 13.3 years) who were admitted to the Molecular Microbiology Unit of Department of Medical Microbiology, Karadeniz Technical University Faculty of Medicine, between January 2009 to December 2012, were included in the study. HCV genotypes were detected in plasma samples of the patients by using commercial assays [INNO-LiPA HCV II (Innogenetics, Belgium) or Abbott RealTime HCV Genotype II (Abbott Molecular Inc, USA)]. Due to the ambiguous genotyping results in some samples with these methods, an in-house multiplex polymerase chain reaction (PCR) assay with genotype-specific primers was also used in the study. Similar to the previous reports from Turkey, our results showed that four HCV genotypes (1, 2, 3, and 4) prevailed in the Eastern Black Sea Region and the predominant genotype and subtype were genotype 1 (92.8%) and 1b (87.5%), respectively. Distribution of genotypes were observed to vary according to the province. Prevalences of subtype 1a, genotype 2, 3, and 4 were noted as 5.3%, 1.6%, 4.9%, and 0.7%, respectively. Furthermore, the samples from Giresun, Gumushane and Bayburt provinces, which are relatively less immigrated, had higher genotype 1, and the prevalence rates in the region was affected by the presence of non-citizen residents. This study is the first report on distribution of HCV genotypes in chronic hepatitis

  18. Regional profiling for determination of genotype diversity of mastitis-specific Staphylococcus aureus lineage in Canada by use of clumping factor A, pulsed-field gel electrophoresis, and spa typing.

    PubMed

    Said, Kamaleldin B; Ismail, Johanne; Campbell, Jennifer; Mulvey, Michael R; Bourgault, Anne-Marie; Messier, Serge; Zhao, Xin

    2010-02-01

    One of the major concerns in global public health and the dairy industry is the emergence of host-specific virulent Staphylococcus aureus strains. The high degree of stability of the species genome renders detection of genetic microvariations difficult. Thus, approaches for the rapid tracking of specialized lineages are urgently needed. We used clumping factor A (clfA) to profile 87 bovine mastitis isolates from four regions in Canada and compared the results to those obtained by pulsed-field gel electrophoresis (PFGE) and spa typing. Twenty-five pulsotypes were obtained by PFGE with an index of discrimination of 0.91. These were assigned to six PFGE lineage groups A to F and seven spa types, including two novel ones. Group A had 48.3% of the isolates and group D had 43.7% of the isolates, while only 8% of the isolates were variable. The results of antimicrobial susceptibility testing indicated that all isolates were sensitive to methicillin and the non-beta-lactam antibiotics, while three isolates were resistant to penicillin and one isolate was resistant to tetracycline. All isolates had the clfA gene and belonged to 20 clfA repeat types with an index of discrimination of 0.90. The dominant clfA types, types X, Q, C, and Z, formed 82% and 43% of PFGE groups A and D, respectively, and had copy numbers that varied only within a narrow range of between 46 and 52 copies, implying clonal selection. The rest were variable and region specific. Furthermore, the dominant groups contained subpopulations in different regions across Canada. Sequence information confirmed the relatedness obtained by the use of clfA repeat copy numbers and other methods and further revealed the occurrence of full-repeat deletions and conserved host-specific codon-triplet position biases at 18-bp units. Thus, concordant with the results of PFGE and spa typing, clfA typing proved useful for revealing the clonal nature of the mastitis isolate lineage and for the rapid profiling of subpopulations

  19. 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

  20. Effects of plant genotype and insect dispersal rate on the population dynamics of a forest pest.

    PubMed

    Moran, Emily V; Bewick, Sharon; Cobbold, Christina A

    2013-12-01

    It has been shown that plant genotype can strongly affect not only individual herbivore performance, but also community composition and ecosystem function. Few studies, however, have addressed how plant genotype affects herbivore population dynamics. In this paper, we used a simulation modeling approach to ask how the genetic composition of a forest influences pest outbreak dynamics, using the example of aspen (Populus tremuloides) and forest tent caterpillars (FTC; Malacosoma disstria). Specifically, we examined how plant genotype, the relative size of genotypic patches, and the rate of insect dispersal between them, affect the frequency, amplitude, and duration of outbreaks. We found that coupling two different genotypes does not necessarily result in an averaging of insect dynamics. Instead, depending on the ratio of patch sizes, when dispersal rates are moderate, outbreaks in the two-genotype case may be more or less severe than in forests of either genotype alone. Thresholds for different dynamic behaviors were similar for all genotypic combinations. Thus, the qualitative behavior of a stand of two different genotypes can be predicted based on the response of the insect to each genotype, the relative sizes of the two patches, and the scale of insect dispersal. PMID:24597225

  1. [DETERMINATION OF GIARDIA DUODENALIS GENOTYPES IN LAMBLIA-INFESTED PEOPLE IN THE CITY OF MOSCOW].

    PubMed

    Kurnosova, O P; Odoevskaya, I M; Krasavchenko, K S

    2015-01-01

    Our first experience in genotyping Giardia from Moscow residents, has shown that 4 and 2 of seven samples belong to G. duodenalis genotype A and genotype B, respectively; one sample was negative during amplification with two types of primers. Genotyping was Carried out using the specific primers TPIA and TPIB to the gene encoding for the enzyme triosephosphate isomerase from the parasite. Thus, further such investigations using a larger number of samples will be able to complement the epidemiology of Lamblia infection in Moscow residents. PMID:26720965

  2. DNA probe for detection of the Leptospira interrogans serovar hardjo genotype hardjo-bovis.

    PubMed Central

    LeFebvre, R B

    1987-01-01

    A DNA probe is described for the diagnostic and taxonomic identification of the North American cattle pathogen Leptospira interrogans genotype hardjo-bovis. The probe is specific for this genotype and does not hybridize to genomic DNA of any other leptospire pathogen commonly found in North America. Images PMID:2826538

  3. Transforming microbial genotyping: a robotic pipeline for genotyping bacterial strains.

    PubMed

    O'Farrell, Brian; Haase, Jana K; Velayudhan, Vimalkumar; Murphy, Ronan A; Achtman, Mark

    2012-01-01

    Microbial genotyping increasingly deals with large numbers of samples, and data are commonly evaluated by unstructured approaches, such as spread-sheets. The efficiency, reliability and throughput of genotyping would benefit from the automation of manual manipulations within the context of sophisticated data storage. We developed a medium- throughput genotyping pipeline for MultiLocus Sequence Typing (MLST) of bacterial pathogens. This pipeline was implemented through a combination of four automated liquid handling systems, a Laboratory Information Management System (LIMS) consisting of a variety of dedicated commercial operating systems and programs, including a Sample Management System, plus numerous Python scripts. All tubes and microwell racks were bar-coded and their locations and status were recorded in the LIMS. We also created a hierarchical set of items that could be used to represent bacterial species, their products and experiments. The LIMS allowed reliable, semi-automated, traceable bacterial genotyping from initial single colony isolation and sub-cultivation through DNA extraction and normalization to PCRs, sequencing and MLST sequence trace evaluation. We also describe robotic sequencing to facilitate cherrypicking of sequence dropouts. This pipeline is user-friendly, with a throughput of 96 strains within 10 working days at a total cost of < €25 per strain. Since developing this pipeline, >200,000 items were processed by two to three people. Our sophisticated automated pipeline can be implemented by a small microbiology group without extensive external support, and provides a general framework for semi-automated bacterial genotyping of large numbers of samples at low cost. PMID:23144721

  4. Transforming Microbial Genotyping: A Robotic Pipeline for Genotyping Bacterial Strains

    PubMed Central

    Velayudhan, Vimalkumar; Murphy, Ronan A.; Achtman, Mark

    2012-01-01

    Microbial genotyping increasingly deals with large numbers of samples, and data are commonly evaluated by unstructured approaches, such as spread-sheets. The efficiency, reliability and throughput of genotyping would benefit from the automation of manual manipulations within the context of sophisticated data storage. We developed a medium- throughput genotyping pipeline for MultiLocus Sequence Typing (MLST) of bacterial pathogens. This pipeline was implemented through a combination of four automated liquid handling systems, a Laboratory Information Management System (LIMS) consisting of a variety of dedicated commercial operating systems and programs, including a Sample Management System, plus numerous Python scripts. All tubes and microwell racks were bar-coded and their locations and status were recorded in the LIMS. We also created a hierarchical set of items that could be used to represent bacterial species, their products and experiments. The LIMS allowed reliable, semi-automated, traceable bacterial genotyping from initial single colony isolation and sub-cultivation through DNA extraction and normalization to PCRs, sequencing and MLST sequence trace evaluation. We also describe robotic sequencing to facilitate cherrypicking of sequence dropouts. This pipeline is user-friendly, with a throughput of 96 strains within 10 working days at a total cost of < €25 per strain. Since developing this pipeline, >200,000 items were processed by two to three people. Our sophisticated automated pipeline can be implemented by a small microbiology group without extensive external support, and provides a general framework for semi-automated bacterial genotyping of large numbers of samples at low cost. PMID:23144721

  5. T-cell response relative to genotype and ethnicity during antiviral therapy for chronic hepatitis C.

    PubMed

    Kaplan, David E; Sugimoto, Kazushi; Ikeda, Fusao; Stadanlick, Jason; Valiga, Mary; Shetty, Kirti; Reddy, K Rajender; Chang, Kyong-Mi

    2005-06-01

    Viral genotype and host ethnicity are important predictors of viral clearance during antiviral therapy for chronic hepatitis C virus (HCV) infection. Based on the role of T cells in natural HCV clearance, we hypothesized that T cells may contribute to the genotypic and ethnic difference in treatment outcome. To test this hypothesis, T-cell response to HCV antigens (core, nonstructural NS3/4 and NS5) and control phytohemagglutinin (PHA) was monitored prospectively and was correlated with virological outcome in 41 patients chronically infected with HCV (27 genotype 1, 14 genotype 2 or 3; 19 black persons, 22 white persons) undergoing combined interferon alfa and ribavirin therapy. Interestingly, in patients with genotype 2 or 3 infection, enhanced virological response coincided with a greater T-cell response to HCV NS3/4 antigen at baseline (50% vs. 15%; P = .026) that augmented further during therapy (29% vs. 4%; P = .035) compared with genotype 1-infected patients. However, HCV-specific T-cell response remained weak in genotype 1-infected patients regardless of virological outcome or ethnicity. Furthermore, virological outcome was associated with a suppressed baseline proliferative response to phytohemagglutinin (P < .03) that increased during therapy (P < .003) independent of ethnicity or genotype. In conclusion, HCV-specific T-cell response was associated with HCV genotype but not with therapeutic clearance of HCV infection. The association between treatment outcome and phytohemagglutinin response suggests more global and antigen-nonspecific mechanisms for therapeutic HCV clearance. PMID:15915458

  6. Polymerase chain reaction-based genotype classification among human Blastocystis hominis populations isolated from different countries.

    PubMed

    Yoshikawa, Hisao; Wu, Zhiliang; Kimata, Isao; Iseki, Motohiro; Ali, Ibne Karim M D; Hossain, Momammad B; Zaman, Viqar; Haque, Rashidul; Takahashi, Yuzo

    2004-01-01

    Since the genotype of human Blastocystis hominis isolates is highly polymorphic, PCR-based genotype classification using known sequenced-tagged site (STS) primers would allow the identification or classification of different genotypes. Five populations of human B. hominis isolates obtained from Japan, Pakistan, Bangladesh, Germany, and Thailand were subjected to genotype analysis by using seven kinds of STS primers. Ninety-nine out of 102 isolates were identified as one of the known genotypes, while one isolate from Thailand showed two distinct genotypes and two isolates from Japan were negative with all the STS primers. The most dominant genotype among four populations, except for all four isolates from Thailand, was subtype 3 and it varied from 41.7% to 92.3%. The second most common genotype among four populations was either subtype 1 (7.7-25.0%) or subtype 4 (10.0-22.9%). Subtype 2, subtype 5, and/or subtype 7 were only rarely detected among the isolates from Japan and Germany, while subtype 6 was not detected. The phylogenetic position of the two isolates which were negative with all STS primers, was inferred from the small subunit rRNA (SSU rRNA) genes with the known sequence data of 20 Blastocystis isolates. Since the two isolates were positioned in an additional clade in the phylogenetic tree, this suggested they were a new genotype. These results demonstrated that PCR-based genotype classification is a powerful tool with which to analyse genotypes of Blastocystis isolates obtained from clinical samples. In addition, two groups of the isolates from 15 symptomatic and 11 asymptomatic patients in Bangladesh were compared with the PCR-based subtype classification. Since both groups were only classified into two distinct genotypes of subtype 1 or subtype 3 and no statistically significant difference was observed between the two groups, in this study it could not be shown that the specific genotype correlated with the pathogenic potential of B. hominis. PMID

  7. 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

  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. Determination of hepatitis C virus genotype by Pyrosequencing.

    PubMed

    Elahi, Elahe; Pourmand, Nader; Chaung, Ramsey; Rofoogaran, Ara; Boisver, Judie; Samimi-Rad, Katayon; Davis, Ronald W; Ronaghi, Mostafa

    2003-05-01

    A simple sequencing-based assay is described for genotyping of hepatitis C virus (HCV). RT-PCR was employed to amplify a 237-nucleotide-long fragment from the 5' untranslated region (UTR) of the genome using one biotinylated and one normal primer. Subsequent to capture of the PCR products on streptavidin-coated beads, single-stranded DNA separation, and hybridization of sequencing primer, Pyrosequencing was performed. The genotype of 98 samples out of which 77 samples were from American veterans and 21 samples were from Iran was determined. The samples from the American veterans contained six different subtypes, while five subtypes were found in Iranian samples. For rapid population-specific HCV subtyping, a multiplex assay was developed. This study demonstrates the suitability of this technology for low-cost, high throughput and accurate microbial genotyping. PMID:12711060

  10. 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

  11. Improved Ancestry Estimation for both Genotyping and Sequencing Data using Projection Procrustes Analysis and Genotype Imputation

    PubMed Central

    Wang, Chaolong; Zhan, Xiaowei; Liang, Liming; Abecasis, Gonçalo R.; Lin, Xihong

    2015-01-01

    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

  12. Impact of Human Enterovirus 71 Genotypes in Meningoencephalitis in Iran

    PubMed Central

    Rahimi, Pooneh; Roohandeh, Akram; Sohrabi, Amir; Mostafavi, Ehsan; Bahram Ali, Golnaz

    2015-01-01

    Background: Since the importance of poliovirus has diminished, as a result of its elimination in the majority of countries, non-polioviruses are emerging as causative agents of severe central nervous system (CNS) involvement. Outbreaks of enterovirus 71 (EV71)-associated CNS infections have recently been reported in Asia, Australia, and Europe. Objectives: This is the first study on genotyping of EV71 in children with meningoencephalitis to be carried out in Iran, and it was conducted in order to obtain an improved understanding of the disease burden of this virus, particularly with regard to CNS involvement. Patients and Methods: Viral RNA was extracted from 170 cerebrospinal fluid samples obtained from children aged under 8 years with a primary diagnosis of aseptic meningitis. Specific EV71 PCR was conducted to identify the genotype of the detected EV71 viruses. Results: Human enteroviruses (HEVs) were detected in 89 patients (52.3%). EV71 infection was detected in 19 (21.3%) of the 89 EV71-positive patients, and the C genotype was identified in 15 isolates. Conclusions: The C genotype should be considered as the prevalent EV71 circulating genotype in Iran, particularly in cases of aseptic meningitis. PMID:26865943

  13. APOE genotype alters immunoglobulin subtypes in knock-in mice

    PubMed Central

    Zhou, Ye; Zhao, Wenjuan; Al-muhtasib, Nour; Rebeck, G. William

    2016-01-01

    Apolipoprotein E (APOE) alleles are strongly related to the risk of Alzheimer’s disease (AD). APOE genotype also affects inflammatory processes in response to damage. We tested whether APOE genotype affected the levels of specific immunoglobulins in healthy, uninfected APOE knock-in mice. We measured specific immunoglobulins in brain, spleen and plasma. Levels of total IgG in brain and spleen were highest in APOE-ε3 mice, significantly higher than in APOE-ε2 and APOE-ε4 mice; no differences were observed for levels of total IgG plasma. We also measured specific subtypes of IgG. IgG1 was only detectable in plasma, and did not differ by APOE genotype. IgG3 was detectable in plasma and spleen, and also did not differ by APOE genotype. IgG2b showed the same pattern as levels of total IgG by APOE genotype, with the highest levels of IgG2b in brain, spleen, and plasma of APOE-ε3 mice. IgG2a showed an entirely different pattern, with significantly higher levels in spleen and plasma of APOE-ε4 mice compared to APOE-ε2 and APOE-ε3 mice. We also measured IgM and IgA in spleens and plasma of these mice. In spleen, APOE-ε4 mice had the lowest IgA levels and the highest levels of IgM; both being significantly different from APOE-ε2 mice. In total, murine IgG2a and IgM were highest in APOE-ε4 mice, while total IgG and Ig2b were highest in APOE-ε3 mice. These dramatically different distributions of immunoglobulins could allow for human AD risk biomarkers based on specific immunoglobulin subtypes. PMID:25737044

  14. A novel method for multiplex genotyping in a single reactor using GTPlex-PyroSeq: genotyping HPV as a prototype.

    PubMed

    Oh, Myungsok; Hoehn, Benjamin Douglass; Moon, Youngho; Oh, Taejeong; Ko, Youngbok; An, Sungwhan

    2012-07-01

    Herein, we describe a novel multiplex genotyping method, GTPlex-PyroSeq. This method consists of two phases: multiplex PCR followed by a single reaction of pyrosequencing. This study demonstrates how GTPlex-PyroSeq can be adapted for the determination of multiple human papillomavirus (HPV) genotypes. A biotinylated consensus primer, GP6+, and 15 high-risk HPV type-specific primers are used for multiplex PCR. Each type-specific primer has a 5'-tag unique ID sequence connected to a pyrosequencing primer binding region. The unique ID sequence is composed of three parts: i) a single nucleotide ID representing a specific genotype; ii) a sign post; and iii) an end mark. This design allows multiple genotype determination under an ID sequence-dependent nucleotide dispensation order during pyrosequencing. Following initial studies using HPV plasmids and cell lines, we evaluated the clinical utility and effectiveness by comparing our assay with direct sequencing and HPV DNA chip analysis of 80 samples from high-risk, HPV-positive patients. We found in single-type infections, 100% concordance with direct sequencing (70 of 80 perfect matches) and 97.5% concordance with HPV DNA chip data (50 of 80 perfect matches). Additionally, our system was superior to direct sequencing in detection of multiple infections (12 of 80), with a limit of detection of 100 copies. The scalability of this multiplex system, with its open-platform design and ability to use various sample types, makes the GTPlex applicable for use in multiple settings. PMID:22575716

  15. 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

  16. 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

  17. 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

  18. 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. PMID:26423670

  19. Current software for genotype imputation.

    PubMed

    Ellinghaus, David; Schreiber, Stefan; Franke, Andre; Nothnagel, Michael

    2009-07-01

    Genotype imputation for single nucleotide polymorphisms (SNPs) has been shown to be a powerful means to include genetic markers in exploratory genetic association studies without having to genotype them, and is becoming a standard procedure. A number of different software programs are available. In our experience, user-friendliness is often the deciding factor in the choice of software to solve a particular task. We therefore evaluated the usability of three publicly available imputation programs: BEAGLE, IMPUTE and MACH. We found all three programs to perform well with HapMap reference data, with little effort needed for data preparation and subsequent association analysis. Each of them has different strengths and weaknesses, however, and none is optimal for all situations. PMID:19706367

  20. 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. PMID:21750470

  1. Rapid and sensitive genotyping of hepatitis C virus by single-strand conformation polymorphism.

    PubMed

    Lareu, R R; Swanson, N R; Fox, S A

    1997-02-01

    There is an increasing demand for genotyping hepatitis C virus (HCV) isolates due to the rapidly expanding list of distinct HCV genotypes and the mounting evidence of genotype-specific clinical consequences. We describe an SSCP-based assay for determining genotypes in HCV infections. HCV RNA extracted from serum was amplified by a sensitive nested-PCR assay producing a 287 bp fragment of the conserved 5' non-coding region (NCR) and analysed by non-denaturing polyacrylamide gel electrophoresis. Following empirical optimisation of the SSCP assay we identified distinct conformation polymorphisms (characteristic band patterns) corresponding to types 1a, 1b, 2a, 2b, 2c, 3 and 4 found in the Western Australian population. Seventy-three HCV RNA-positive samples were used to evaluate the SSCP genotyping assay for accuracy and efficiency by comparison with the previously established genotyping methods of manual direct sequencing and dideoxy fingerprinting. SSCP genotyping was in concord with control methods while performing more rapidly and at a fraction of the cost. Moreover, SSCP detected two co-infected samples that were not shown by the control methods. The PCR-SSCP assay provides an accurate and rapid method for genotyping of HCV RNA-positive samples at the 5' NCR by type-specific sequence polymorphisms which is applicable to large-scale screening. PMID:9029525

  2. Histoimmunogenetics Markup Language 1.0: Reporting next generation sequencing-based HLA and KIR genotyping.

    PubMed

    Milius, Robert P; Heuer, Michael; Valiga, Daniel; Doroschak, Kathryn J; Kennedy, Caleb J; Bolon, Yung-Tsi; Schneider, Joel; Pollack, Jane; Kim, Hwa Ran; Cereb, Nezih; Hollenbach, Jill A; Mack, Steven J; Maiers, Martin

    2015-12-01

    We present an electronic format for exchanging data for HLA and KIR genotyping with extensions for next-generation sequencing (NGS). This format addresses NGS data exchange by refining the Histoimmunogenetics Markup Language (HML) to conform to the proposed Minimum Information for Reporting Immunogenomic NGS Genotyping (MIRING) reporting guidelines (miring.immunogenomics.org). Our refinements of HML include two major additions. First, NGS is supported by new XML structures to capture additional NGS data and metadata required to produce a genotyping result, including analysis-dependent (dynamic) and method-dependent (static) components. A full genotype, consensus sequence, and the surrounding metadata are included directly, while the raw sequence reads and platform documentation are externally referenced. Second, genotype ambiguity is fully represented by integrating Genotype List Strings, which use a hierarchical set of delimiters to represent allele and genotype ambiguity in a complete and accurate fashion. HML also continues to enable the transmission of legacy methods (e.g. site-specific oligonucleotide, sequence-specific priming, and Sequence Based Typing (SBT)), adding features such as allowing multiple group-specific sequencing primers, and fully leveraging techniques that combine multiple methods to obtain a single result, such as SBT integrated with NGS. PMID:26319908

  3. Prevalence of HCV genotypes in district Mardan

    PubMed Central

    2013-01-01

    Background Approximately 170 million people are infected with Hepatitis C virus (HCV) worldwide. The prevalence of chronic HCV infections in Pakistan is about 5%, with most individuals being infected with HCV genotype 3a. Data on HCV genotypes distribution across various districts of the country are scarce. One example is district Mardan from where such data is available only from 17 individuals. Accordingly, the present study aimed at determining HCV genotypes distribution among 177 HCV RNA positive individuals from district Mardan. Findings Serum samples (n = 215) from patients suspected of hepatitis C were collected and processed for Nested PCR based detection and subsequent genotyping. Gender-wise and age-wise differences in HCV prevalence and HCV genotypes distribution were determined by χ2 test. Out of the total 215 serum samples, 177 were found to be positive for HCV RNA. The genotype 3a was the most predominant genotype among HCV RNA positive samples with a prevalence of 90.3%, followed by genotype 1a (5.6%), mixed genotypes (2.8%), genotype 3b (0.6%) and genotype 4 (0.6%). The HCV prevalence was higher in young individuals than old people and was indicative of reduced survival rate beyond 40 years. Conclusion HCV genotype 3a is the most predominant genotype in district Mardan. The state of the art preventive and therapeutic strategies should be implemented to control the spread of HCV infections. Further temporal studies involving different geographical areas of Pakistan, are required to improve the control measures for HCV infection. PMID:23514695

  4. 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

  5. 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. PMID:27242776

  6. 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

  7. Hepatitis C virus genotypes in north eastern Algeria: A retrospective study

    PubMed Central

    Rouabhia, Samir; Sadelaoud, Mourad; Chaabna-Mokrane, Karima; Toumi, Wided; Abenavoli, Ludovico

    2013-01-01

    AIM: To determine the frequency of various hepatitis C virus (HCV) genotypes present in patients from north eastern Algeria. METHODS: This is a retrospective cross-sectional study of 435 HCV infected patients from northeast Algeria, detected in the Sadelaoud laboratory and diagnosed between January 2010 and December 2012. The patients were diagnosed with HCV infection in their local hospitals and referred to be assessed for HCV genotype before the antiviral treatment. Demographic information (sex, age and address), genotype, subtype and viral load were retrieved from the patient medical records. The serum samples were tested by the type-specific genotyping assay. RESULTS: The majority of the patients (82.5%) were from the central part of the examined region (P = 0.002). The mean age of the patients studied was 53.6 ± 11.5 years. HCV genotype 1 was the most frequent (88.7%), followed by genotypes 2 (8.5%), 4 (1.1%), 3 (0.9%) and 5 (0.2%). Genotype 6 was not detected in these patients. Mixed infection across the HCV subtypes was detected in twenty patients (4.6%). The genotype distribution was related to age and region. Genotype 1 was significantly less frequent in the ≥ 60 age group than in the younger age group (OR = 0.2; 95%CI: 0.1-0.5, P < 0.001). Furthermore, genotype 1 was more frequent in the central part of the examined region than elsewhere (P < 0.01). CONCLUSION: The HCV genotype (type 1b was dominant) distribution in Algeria is different from those in other northern countries of Africa. PMID:23898373

  8. Heterogeneous Host Susceptibility Enhances Prevalence of Mixed-Genotype Micro-Parasite Infections

    PubMed Central

    Vlak, Just M.; Zwart, Mark P.

    2011-01-01

    Dose response in micro-parasite infections is usually shallower than predicted by the independent action model, which assumes that each infectious unit has a probability of infection that is independent of the presence of other infectious units. Moreover, the prevalence of mixed-genotype infections was greater than predicted by this model. No probabilistic infection model has been proposed to account for the higher prevalence of mixed-genotype infections. We use model selection within a set of four alternative models to explain high prevalence of mixed-genotype infections in combination with a shallow dose response. These models contrast dependent versus independent action of micro-parasite infectious units, and homogeneous versus heterogeneous host susceptibility. We specifically consider a situation in which genome differences between genotypes are minimal, and highly unlikely to result in genotype-genotype interactions. Data on dose response and mixed-genotype infection prevalence were collected by challenging fifth instar Spodoptera exigua larvae with two genotypes of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), differing only in a 100 bp PCR marker sequence. We show that an independent action model that includes heterogeneity in host susceptibility can explain both the shallow dose response and the high prevalence of mixed-genotype infections. Theoretical results indicate that variation in host susceptibility is inextricably linked to increased prevalence of mixed-genotype infections. We have shown, to our knowledge for the first time, how heterogeneity in host susceptibility affects mixed-genotype infection prevalence. No evidence was found that virions operate dependently. While it has been recognized that heterogeneity in host susceptibility must be included in models of micro-parasite transmission and epidemiology to account for dose response, here we show that heterogeneity in susceptibility is also a fundamental principle explaining

  9. Two distinct human parainfluenza virus type 1 genotypes detected during the 1991 Milwaukee epidemic.

    PubMed Central

    Henrickson, K J; Savatski, L L

    1996-01-01

    The extent of genetic and antigenic variation found in a population of human parainfluenza virus type 1 (HPIV-1) during a single local epidemic was investigated. Fifteen HPIV-1 strains isolated from children in 1991 were analyzed. Nucleotide sequence variation in the hemagglutinin-neuraminidase protein (HN) gene demonstrated two distinct genotypes (genotypes C and D). Unique patterns were identified involving 62 nucleotide and 10 amino acid positions. These patterns represented 40% of all mutations within the HN gene. The remaining mutations were randomly distributed, and 74% involved only one (55%) or two isolates. Genotypes were statistically different from each other at both the nucleotide (P = 0.001) and amino acid (P = 0.001) levels and demonstrated unique potential N-linked glycosylation patterns. Thirty-eight monoclonal antibodies (MAbs) made to four different viral proteins (22 HN, 2 fusion [F], 1 phosphoprotein, and 13 nucleoprotein) (originating from two different genotypes [genotypes A and D]) were compared for their ability to bind to the clinical isolates in enzyme-linked immunosorbent assays (ELISAs) and hemagglutinin-inhibition (HI) assays. Twenty-one MAbs bound well to all clinical isolates in ELISAs and HI assays. The remaining 17 MAbs showed variation in all four structural proteins. Three HN MAbs demonstrated genotype C- and D-specific antigenic and neutralization differences. Evolutionary analysis using parsimony methods confirmed the differences between the two genotypes. No differences in either clinical presentation or disease severity between the two genotypes were found. Geographically localized HPIV-1 epidemics can be caused by at least two distinct genotypes with minor but specific antigenic changes. The clinical and immunologic roles of HPIV-1 genotypes have not been determined. PMID:8904440

  10. 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

  11. 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... affect the usability of the facility or part of the facility shall to the maximum extent feasible, be... equivalent or greater access to and usability of the building is provided. (2) For purposes of this...

  12. Human parvovirus (B19) and erythema infectiosum.

    PubMed

    Nunoue, T; Okochi, K; Mortimer, P P; Cohen, B J

    1985-07-01

    An outbreak of erythema infectiosum ("fifth disease") was studied in Fukuoka, Japan, in 1980-1981. Human parvovirus (HPV) antigen was not detected in any patients, but anti-HPV, measured by countercurrent immunoelectrophoresis, was found in 33 of 34 affected children and in 21 (15%) of 141 children of the same ages without the disease. Immunoglobulin M class anti-HPV was present in all 25 children with erythema infectiosum tested. In a survey of hospital patients, the prevalence of anti-HPV detected by CIE was 12% in the cohort 5 to 9 years of age, 19% in the cohort 10 to 14 years, and 32 to 55% in the cohorts greater than or equal to 30 years. The antibody reactions in the cases of erythema infectiosum, which were already well established at the onset of disease, indicate that HPV was the cause of the outbreak. PMID:2989471

  13. Rotavirus Genotypes in Belarus, 2008–2012

    PubMed Central

    Semeiko, Galina; Yermalovich, Marina; Poliakova, Nadezhda; Mijatovic-Rustempasic, Slavica; Kerin, Tara; Wasley, Annemarie; Videbaek, Dovile; Gentsch, Jon R.; Bowen, Michael D.; Samoilovich, Elena

    2015-01-01

    This study describes group A rotavirus (RVA) genotype prevalence in Belarus from 2008–2012. 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. PMID:25218086

  14. 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

  15. Genotype-based databases for variants causing rare diseases.

    PubMed

    Lanthaler, Barbara; Wieser, Stefanie; Deutschmann, Andrea; Schossig, Anna; Fauth, Christine; Zschocke, Johannes; Witsch-Baumgartner, Martina

    2014-10-15

    Inherited diseases are the result of DNA sequence changes. In recessive diseases, the clinical phenotype results from the combined functional effects of variants in both copies of the gene. In some diseases there is often considerable variability of clinical presentation or disease severity, which may be predicted by the genotype. Additional effects may be triggered by environmental factors, as well as genetic modifiers which could be nucleotide polymorphisms in related genes, e.g. maternal ApoE or ABCA1 genotypes which may have an influence on the phenotype of SLOS individuals. Here we report the establishment of genotype variation databases for various rare diseases which provide individual clinical phenotypes associated with genotypes and include data about possible genetic modifiers. These databases aim to be an easy public access to information on rare and private variants with clinical data, which will facilitate the interpretation of genetic variants. The created databases include ACAD8 (isobutyryl-CoA dehydrogenase deficiency (IBD)), ACADSB (short-chain acyl-CoA dehydrogenase (SCAD) deficiency), AUH (3-methylglutaconic aciduria (3-MGCA)), DHCR7 (Smith-Lemli-Opitz syndrome), HMGCS2 (3-hydroxy-3-methylglutaryl-CoA synthase 2 deficiency), HSD17B10 (17-beta-hydroxysteroid dehydrogenase X deficiency), FKBP14 (Ehlers-Danlos syndrome with progressive kyphoscoliosis, myopathy, and hearing loss; EDSKMH) and ROGDI (Kohlschütter-Tönz syndrome). These genes have been selected because of our specific research interests in these rare and metabolic diseases. The aim of the database was to include all identified individuals with variants in these specific genes. Identical genotypes are listed multiple times if they were found in several patients, phenotypic descriptions and biochemical data are included as detailed as possible in view also of validating the proposed pathogenicity of these genotypes. For DHCR7 genetic modifier data (maternal APOE and ABCA1 genotypes) is

  16. 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

  17. A MEMS-Based Approach to Single Nucleotide Polymorphism Genotyping

    PubMed Central

    Zhu, Jing; Palla, Mirkó; Ronca, Stefano; Warpner, Ronald; Ju, Jingyue; Lin, Qiao

    2014-01-01

    Genotyping of single nucleotide polymorphisms (SNPs) allows diagnosis of human genetic disorders associated with single base mutations. Conventional SNP genotyping methods are capable of providing either accurate or high-throughput detection, but are still labor-, time-, and resource-intensive. Microfluidics has been applied to SNP detection to provide fast, low-cost, and automated alternatives, although these applications are still limited by either accuracy or throughput issues. To address this challenge, we present a MEMS-based SNP genotyping approach that uses solid-phase-based reactions in a single microchamber on a temperature control chip. Polymerase chain reaction (PCR), allele specific single base extension (SBE), and desalting on microbeads are performed in the microchamber, which is coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze the SBE product. Experimental results from genotyping of the SNP on exon 1 of the HBB gene, which causes sickle cell anemia, demonstrate the potential of the device for rapid, accurate, multiplexed and high-throughput detection of SNPs. PMID:24729659

  18. A Microfluidic Device for Multiplex Single-Nucleotide Polymorphism Genotyping

    PubMed Central

    Zhu, Jing; Qiu, Chunmei; Palla, Mirkó; Nguyen, ThaiHuu; Russo, James J.; Ju, Jingyue; Lin, Qiao

    2015-01-01

    Single-nucleotide polymorphisms (SNPs) are the most abundant type of genetic variations; they provide the genetic fingerprint of individuals and are essential for genetic biomarker discoveries. Accurate detection of SNPs is of great significance for disease prevention, diagnosis and prognosis, and for prediction of drug response and clinical outcomes in patients. Nevertheless, conventional SNP genotyping methods are still limited by insufficient accuracy or labor-, time-, and resource-intensive procedures. Microfluidics has been increasingly utilized to improve efficiency; however, the currently available microfluidic genotyping systems still have shortcomings in accuracy, sensitivity, throughput and multiplexing capability. To address these challenges, we developed a multi-step SNP genotyping microfluidic device, which performs single-base extension of SNP specific primers and solid-phase purification of the extension products on a temperature-controlled chip. The products are ready for immediate detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), providing identification of the alleles at the target loci. The integrated device enables efficient and automated operation, while maintaining the high accuracy and sensitivity provided by MS. The multiplex genotyping capability was validated by performing rapid, accurate and simultaneous detection of 4 loci on a synthetic template. The microfluidic device has the potential to perform automatic, accurate, quantitative and high-throughput assays covering a broad spectrum of applications in biological and clinical research, drug development and forensics. PMID:26594354

  19. 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

  20. Hepatitis B virus: the genotype E puzzle.

    PubMed

    Andernach, Iris E; Hübschen, Judith M; Muller, Claude P

    2009-07-01

    Hepatitis B virus (HBV) is highly endemic throughout sub-Saharan Africa. One of the two genotypes A and E dominates in most countries. With several subgenotypes and variants, genotype A is more diverse in Africa (4.00%) than in the rest of the world (2.96%), suggesting an African origin and a long history on the continent. Despite the African slave trade, genotype E has only sporadically been found within the Americas, indicating that this genotype was introduced only during the past 200 years into the general African population. A short history for this genotype in Africa is also supported by its conspicuously low genetic diversity (1.75%), which contrasts, however, with its excessively high HBsAg prevalence and its extensive spread throughout the vast West-African genotype E crescent. We discuss the spread and routes of transmission of genotype E and suggest that the distribution and current high prevalence levels of HBV (genotype E) in Africa are the result of the extensive use of unsafe needles, potentially solving the current African genotype E puzzle and shedding new light on the high HBV prevalence in Africa. PMID:19475565

  1. 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

  2. Differences in CYP2C9 Genotype and Enzyme Activity Between Swedes and Koreans of Relevance for Personalized Medicine: Role of Ethnicity, Genotype, Smoking, Age, and Sex.

    PubMed

    Hatta, Fazleen H M; Lundblad, Mia; Ramsjo, Margareta; Kang, Ju-Hee; Roh, Hyung-Keun; Bertilsson, Leif; Eliasson, Erik; Aklillu, Eleni

    2015-06-01

    Global personalized medicine demands the characterization of person-to-person and between-population differences in drug pharmacokinetics and pharmacodynamics. CYP2C9 pharmacokinetic pathway is subject to modulation by both genetic and environmental factors. CYP2C9 genotype-based dose recommendations (e.g., for warfarin) is advocated. However, the overall contribution of genotype for variation in enzyme activity may differ between populations. We evaluated the importance of ethnicity, genotype, smoking, body weight, age, and sex for CYP2C9 enzyme activity. CYP2C9 genotype and phenotype was determined in 148 Swedes and 146 Koreans using losartan as a probe. CYP2C9 enzyme activity was assessed using urinary losartan/metabolite E-3174 ratio. The frequency of CYP2C9 defective variant alleles (*2 and *3) was significantly higher in Swedes (10.8% and 12.5%) than in Koreans (0% and 5.8%). In matched genotypes, CYP2C9 enzyme activity was significantly lower in Swedes compared to Koreans (p<0.0001). In a univariate analysis, age, weight, ethnicity, genotype, and smoking were significant predictors of CYP2C9 phenotype. A stepwise multivariate analysis indicated ethnicity, genotype, and smoking remained as significant predictors of CYP2C9 enzyme activity, accounting for 50% of the total variance. In both study populations, CYP2C9 genotype was a significant predictor of CYP2C9 enzyme activity, but its contribution in explaining the total variance was lower in Koreans (26.6%) than Swedes (40%). In conclusion, we report significantly lower CYP2C9 enzyme activity in Swedes compared to Koreans, partly but not exclusively due to CYP2C9 pharmacogenetic variations. Ethnicity and environment factors need to be considered together with genotype for population-specific dose optimization and global personalized medicine. PMID:25977991

  3. Enterocytozoon bieneusi genotypes in dairy cattle in the eastern United States.

    PubMed

    Santín, M; Trout, J M; Fayer, R

    2005-12-01

    Fecal specimens were obtained from 12-24-month-old dairy heifers on farms in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. PCR positive specimens for Enterocytozoon bieneusi were found in 131 of 571 heifers examined (23%) and on all the farms visited. The prevalence of E. bieneusi varied considerably across farms, with the lowest prevalence (4.7%) on MD-2 and the highest prevalence (37.8%) on NY-2. All PCR positive specimens that amplified the ITS region as well as a portion of the flanking large and small subunit ribosomal RNA genes were sequenced to determine the genotype(s) of the E. bieneusi present and six genotypes were identified. Most were identified as cattle-specific genotypes, previously reported from cattle as BEB1, BEB2, BEB3, and BEB4. Two isolates were genetically identical or similar to E. bienesusi reported as the human pathogens Peru 6 and Peru 9 (or D) genotypes. Although our data demonstrate the presence of zoonotic genotypes in cattle, most genotypes found in cattle were host specific. PMID:16167161

  4. Distribution of HPV genotypes in Shanghai women

    PubMed Central

    Singh, Suman; Zhou, Qian; Yu, Yunyun; Xu, Xianghong; Huang, Xiaojie; Zhao, Junwei; Han, Lingfei; Wang, Kai; Sun, Jing; Li, Fang

    2015-01-01

    Objective: To study the Distribution of HPV genotypes in Shanghai women. Design: Cohort study. Setting: Shanghai First Maternity and Infant Hospital affiliated with Tongji University. Population: Patients those attended in the cervical disease diagnosis and treatment center of Shanghai First Maternity and Infant Hospital between January 2011 and December 2014. Methods: HPV GenoArray test kit (HybriBio Ltd) was used to perform HPV genotyping and was also used in DNA amplification and HybriBio’s proprietary flow-through hybridization technique. Results: In this study, total patients analyzed were 4585. Among 4585 sample the HPV positive patients were 1460 i.e. 31.84% in total. On the basis of pathological report normal were 1358, with inflammation 2441, with low grade lesion were 399, high grade lesion were 353, CIN were 19 and cervical carcinoma were 15. Among normal HPV positive were 215 (15.8%), among inflammation HPV positive were 735 (30.11%). HPV positive in low grade lesion were 353 i.e. 59.77%. In high grade lesion 211 were HPV positive among 272 (68.17%). The percentage of HPV positive was 73.68% i.e. 14 out of 19 patient in cervical carcinoma in situ. 13 patient out of 15 i.e. 86.67% of Cervical carcinoma were HPV positive. Among all percentage of HPV positive was high among cervical carcinoma then cervical carcinoma in situ then high grade lesion in decreasing fashion to low grade lesion and in normal. Highest prevalence i.e. 22.67% is of HPV 52 subtype and HPV 16 has second highest prevalence with 17.67% among HPV positive cases. Sensitivity of TCT detection is 71.6%. Specificity of TCT detection is 79.6%. Sensitivity of HPV-DNA detection is 65.2%. Specificity of HPV-DNA detection is 78.2%. Conclusion: HPV is one of major health concern in shanghai having high prevalence rate in comparison to other part of china and other part of world. This has implications for the future cervical cancer burden and the priority to be given to prevent cervical cancer in

  5. Evaluation of the Abbott Real Time HCV genotype II assay for Hepatitis C virus genotyping

    PubMed Central

    Sariguzel, Fatma Mutlu; Berk, Elife; Gokahmetoglu, Selma; Ercal, Baris Derya; Celik, Ilhami

    2015-01-01

    Objective: The determination of HCV genotypes and subtypes is very important for the selection of antiviral therapy and epidemiological studies. The aim of this study was to evaluate the performance of Abbott Real Time HCV Genotype II assay in HCV genotyping of HCV infected patients in Kayseri, Turkey. Methods: One hundred patients with chronic hepatitis C admitted to our hospital were evaluated between June 2012 and December 2012, HCV RNA levels were determined by the COBAS® AmpliPrep/COBAS® TaqMan® 48 HCV test. HCV genotyping was investigated by the Abbott Real Time HCV Genotype II assay. With the exception of genotype 1, subtypes of HCV genotypes could not be determined by Abbott assay. Sequencing analysis was used as the reference method. Results: Genotypes 1, 2, 3 and 4 were observed in 70, 4, 2 and 24 of the 100 patients, respectively, by two methods. The concordance between the two systems to determine HCV major genotypes was 100%. Of 70 patients with genotype 1, 66 showed infection with subtype 1b and 4 with subtype 1a by Abbott Real Time HCV Genotype II assay. Using sequence analysis, 61 showed infection with subtype 1b and 9 with subtype 1a. In determining of HCV genotype 1 subtypes, the difference between the two methods was not statistically significant (P>0.05). HCV genotype 4 and 3 samples were found to be subtype 4d and 3a, respectively, by sequence analysis. There were four patients with genotype 2. Sequence analysis revealed that two of these patients had type 2a and the other two had type 2b. Conclusion: The Abbott Real Time HCV Genotype II assay yielded results consistent with sequence analysis. However, further optimization of the Abbott Real Time HCV Genotype II assay for subtype identification of HCV is required. PMID:26649001

  6. [Application of the PCR-APLP method to determine ABO genotypes in forensic samples].

    PubMed

    Watanabe, G; Umetsu, K; Osawa, M

    2000-08-01

    We carried out ABO genotyping of forensic samples by the amplified product length polymorphism (APLP) technique. We present two novel systems. One is termed as eight primers system, in which eight allele-specific primers are added into a single PCR reaction. Another is termed as six primers system. In both APLP systems, all alleles were clearly detected using DNA purified from forensic samples. In PCR amplification with direct addition of specimen, ABO genotyping was also possible to blood stain, seminal stain, blood, saliva and urine. Furthermore, ABO genotyping worked only to chimpanzee. This PCR-APLP method should be convepffnt and valuable for forensic practice. PMID:11060991

  7. Biological variation in Trichinella species and genotypes.

    PubMed

    Bolas-Fernández, F

    2003-06-01

    At present, the genus Trichinella comprises seven species of which five have encapsulated muscle larvae (T. spiralis, T. nativa, T. britovi, T. nelsoni and T. murrelli) and two do not (T. pseudospiralis and T. papuae) plus three genotypes of non-specific status (T6, T8 and T9). The diagnostic characteristics of these species are based on biological, biochemical and genetic criteria. Of biological significance is variation observed among species and isolates in parameters such as infectivity and immunogenicity. Infectivity of Trichinella species or isolates is determined, among other considerations, by the immune status of the host in response to species- or isolate-specific antigens. Common and particular antigens determine the extent of protective responses against homologous or heterologous challenge. The kinetics of isotype, cytokine and inflammatory responses against T. spiralis infections are isolate-dependent. Trichinella spiralis and T. pseudospiralis induce different dose-dependent T-cell polarizations in the early host response, with T. spiralis initially preferentially promoting Th1-type responses before switching to Th2 and T. pseudospiralis driving Th2-type responses from the outset. PMID:12756064

  8. Geographically predominant genotypes of Aspergillus terreus species complex in Austria: s microsatellite typing study.

    PubMed

    Lackner, M; Coassin, S; Haun, M; Binder, U; Kronenberg, F; Haas, H; Jank, M; Maurer, E; Meis, J F; Hagen, F; Lass-Flörl, C

    2016-03-01

    Aspergillus terreus species complex is recognized as a frequent agent of invasive aspergillosis in Tyrol. The reason for this specific epidemiological situation is unclear. Aspergillus terreus strains isolated from environmental and clinical sources were genotyped using a novel panel of short tandem repeats and were evaluated for virulence. Three major endemic genotypes collected from the Inn region and its side valleys were found to cause the majority of invasive A. terreus infections. All of these genotypes were of the same mating type, which suggests that a mating barrier is present between these geographically well-adapted strains which is found to persist for at least 11 years. The three major genotypes were prevalent in both human infections and the environment. No major differences in virulence were observed using Galleria mellonella as model. Our data suggest a specific environmental exposure being responsible for the high incidence of A. terreus infections in Innsbruck, the Inn valley and side valleys (Tyrol, Austria). PMID:26577144

  9. Human Papillomavirus (HPV) Genotyping: Automation and Application in Routine Laboratory Testing

    PubMed Central

    Torres, M; Fraile, L; Echevarria, JM; Hernandez Novoa, B; Ortiz, M

    2012-01-01

    A large number of assays designed for genotyping human papillomaviruses (HPV) have been developed in the last years. They perform within a wide range of analytical sensitivity and specificity values for the different viral types, and are used either for diagnosis, epidemiological studies, evaluation of vaccines and implementing and monitoring of vaccination programs. Methods for specific genotyping of HPV-16 and HPV-18 are also useful for the prevention of cervical cancer in screening programs. Some commercial tests are, in addition, fully or partially automated. Automation of HPV genotyping presents advantages such as the simplicity of the testing procedure for the operator, the ability to process a large number of samples in a short time, and the reduction of human errors from manual operations, allowing a better quality assurance and a reduction of cost. The present review collects information about the current HPV genotyping tests, with special attention to practical aspects influencing their use in clinical laboratories. PMID:23248734

  10. Human Papillomavirus (HPV) Genotyping: Automation and Application in Routine Laboratory Testing.

    PubMed

    Torres, M; Fraile, L; Echevarria, Jm; Hernandez Novoa, B; Ortiz, M

    2012-01-01

    A large number of assays designed for genotyping human papillomaviruses (HPV) have been developed in the last years. They perform within a wide range of analytical sensitivity and specificity values for the different viral types, and are used either for diagnosis, epidemiological studies, evaluation of vaccines and implementing and monitoring of vaccination programs. Methods for specific genotyping of HPV-16 and HPV-18 are also useful for the prevention of cervical cancer in screening programs. Some commercial tests are, in addition, fully or partially automated. Automation of HPV genotyping presents advantages such as the simplicity of the testing procedure for the operator, the ability to process a large number of samples in a short time, and the reduction of human errors from manual operations, allowing a better quality assurance and a reduction of cost. The present review collects information about the current HPV genotyping tests, with special attention to practical aspects influencing their use in clinical laboratories. PMID:23248734

  11. 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

  12. The E1B19K-deleted oncolytic adenovirus mutant AdΔ19K sensitizes pancreatic cancer cells to drug-induced DNA-damage by down-regulating Claspin and Mre11.

    PubMed

    Pantelidou, Constantia; Cherubini, Gioia; Lemoine, Nick R; Halldén, Gunnel

    2016-03-29

    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

  13. 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.

  14. 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. PMID:25373754

  15. 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

  16. 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. PMID:26377960

  17. 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...

  18. 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 ...

  19. Genotype imputation efficiency in Nelore Cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genotype imputation efficiency in Nelore cattle was evaluated in different scenarios of lower density (LD) chips, imputation methods and sets of animals to have their genotypes imputed. Twelve commercial and virtual custom LD chips with densities varying from 7K to 75K SNPs were tested. Customized L...

  20. Genomic evaluations with many more genotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background Genomic evaluations have quickly become more reliable over the last two years in many countries as more animals were genotyped for 50,000 markers. Evaluations can also include animals genotyped with more or fewer markers using new tools such as 777,000 or 2,900 marker chips recently intr...

  1. Variation in Stress Resistance Patterns among stx Genotypes and Genetic Lineages of Shiga Toxin-Producing Escherichia coli O157

    PubMed Central

    Lee, Ken-ichi; French, Nigel P.; Jones, Geoff; Hara-Kudo, Yukiko; Iyoda, Sunao; Kobayashi, Hideki; Sugita-Konishi, Yoshiko; Tsubone, Hirokazu

    2012-01-01

    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 stx1 stx2 genotype showed greater resistance to heat than strains of the stx1 stx2c genotype; moreover, strains of the stx1 stx2 genotype showed greater resistance to starvation than strains of the stx2 or stx2c 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 stx1 stx2 genotype were grouped with the stress-resistant cluster, whereas 72.7% (8/11) of strains of the stx1 stx2c 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 stx1 stx2 genotype, have greater multiple stress resistance than do strains of other genotypes. PMID:22367077

  2. 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

  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. Parallel genotypic adaptation: when evolution repeats itself

    PubMed Central

    Wood, Troy E.; Burke, John M.; Rieseberg, Loren H.

    2008-01-01

    Until recently, parallel genotypic adaptation was considered unlikely because phenotypic differences were thought to be controlled by many genes. There is increasing evidence, however, that phenotypic variation sometimes has a simple genetic basis and that parallel adaptation at the genotypic level may be more frequent than previously believed. Here, we review evidence for parallel genotypic adaptation derived from a survey of the experimental evolution, phylogenetic, and quantitative genetic literature. The most convincing evidence of parallel genotypic adaptation comes from artificial selection experiments involving microbial populations. In some experiments, up to half of the nucleotide substitutions found in independent lineages under uniform selection are the same. Phylogenetic studies provide a means for studying parallel genotypic adaptation in non-experimental systems, but conclusive evidence may be difficult to obtain because homoplasy can arise for other reasons. Nonetheless, phylogenetic approaches have provided evidence of parallel genotypic adaptation across all taxonomic levels, not just microbes. Quantitative genetic approaches also suggest parallel genotypic evolution across both closely and distantly related taxa, but it is important to note that this approach cannot distinguish between parallel changes at homologous loci versus convergent changes at closely linked non-homologous loci. The finding that parallel genotypic adaptation appears to be frequent and occurs at all taxonomic levels has important implications for phylogenetic and evolutionary studies. With respect to phylogenetic analyses, parallel genotypic changes, if common, may result in faulty estimates of phylogenetic relationships. From an evolutionary perspective, the occurrence of parallel genotypic adaptation provides increasing support for determinism in evolution and may provide a partial explanation for how species with low levels of gene flow are held together. PMID:15881688

  5. The broad assessment of HCV genotypes 1 and 3 antigenic targets reveals limited cross-reactivity with implications for vaccine design

    PubMed Central

    Humphreys, Isla S; Brown, Anthony; Pfafferott, Katja; Lucas, Michaela; Klenerman, Paul; Lauer, Georg M; Cox, Andrea L; Gaudieri, Silvana; Barnes, Eleanor

    2016-01-01

    Objective Developing a vaccine that is cross-reactive between HCV genotypes requires data on T cell antigenic targets that extends beyond genotype-1. We characterised T cell immune responses against HCV genotype-3, the most common infecting genotype in the UK and Asia, and assessed within genotype and between genotype cross-reactivity. Design T cell targets were identified in 140 subjects with either acute, chronic or spontaneously resolved HCV genotype-3 infection using (1) overlapping peptides and (2) putative human leucocyte antigens (HLA)-class-I wild type and variant epitopes through the prior assessment of polymorphic HCV genomic sites associated with host HLA, in IFNγ-ELISpot assays. CD4+/CD8+ T cell subsets were defined and viral variability at T cell targets was determined through population analysis and viral sequencing. T cell cross-reactivity between genotype-1 and genotype-3 variants was assessed. Results In resolved genotype-3 infection, T cells preferentially targeted non-structural proteins at a high magnitude, whereas in chronic disease T cells were absent or skewed to target structural proteins. Additional responses to wild type but not variant HLA predicted peptides were defined. Major sequence viral variability was observed within genotype-3 and between genotypes 1 and 3 HCV at T cell targets in resolved infection and at dominant epitopes, with limited T cell cross-reactivity between viral variants. Overall 41 CD4/CD8+ genotype-3 T cell targets were identified with minimal overlap with those described for HCV genotype-1. Conclusions HCV T cell specificity is distinct between genotypes with limited T cell cross-reactivity in resolved and chronic disease. Therefore, viral regions targeted in natural HCV infection may not serve as attractive targets for a vaccine that aims to protect against multiple HCV genotypes. PMID:26092843

  6. EVALUATION OF GENOTYPIC DIVERSITY OF Streptococcus mutans USING DISTINCT ARBITRARY PRIMERS

    PubMed Central

    Tabchoury, Cínthia Pereira Machado; Sousa, Maria Clara K.; Arthur, Rodrigo Alex; Mattos-Graner, Renata Oliveira; Cury, Altair Antoninha Del Bel; Cury, Jaime Aparecido

    2008-01-01

    Streptococcus mutans has been considered one of the main etiological agents of dental caries and the genotypic diversity rather than its salivary counts may be considered as a virulence factor of this bacterium. For genotyping with polymerase chain reaction (PCR) with arbitrary primers, several primers have been used in order to improve complexity and specificity of amplicon patterns. Thus, the aim of this study was to evaluate the degree of agreement of genotypic identification among AP-PCR reactions performed with 5 distinct arbitrary primers of S. mutans isolated from saliva. Stimulated saliva was collected from 11 adult volunteers for isolation of S. mutans, and a total of 88 isolates were genotyped with arbitrary primers OPA 02, 03, 05, 13 and 18. Fourteen distinct genotypes were identified in the saliva samples. Most volunteers (9 out of 11) presented only one genotype. The results of the present study suggest that primers OPA 02, 03, 05 and 13 were suitable for genotypic identification of S. mutans isolates of saliva from adult volunteers. PMID:19082399

  7. 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

  8. 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

  9. 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. PMID:26973878

  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). PMID:16719392

  12. GABRA2 Genotype, Impulsivity, and Body Mass

    PubMed Central

    Bauer, Lance O.; Yang, Bao-Zhu; Houston, Rebecca J.; Kranzler, Henry R.; Gelernter, Joel

    2013-01-01

    Background The goal of this study was to test a hypothesis associating impulsivity with an elevated body mass index (BMI). Methods To this end, we examined associations of BMI with putative genetic, neurophysiological, psychiatric, and psychological indicators of impulsivity in 78 women and 74 men formerly dependent on alcohol or drugs. A second analysis was designed to test the replicability of the genetic findings in an independent sample of 109 women and 111 men with a similar history of substance dependence. Results The results of the first analysis showed that BMI was positively correlated with Total and Nonplanning Scale Scores on the Barratt Impulsiveness Scale and the number of childhood symptoms of Attention-Deficit/Hyperactivity Disorder in women. It was also positively correlated, in women, with a GABRA2 variant previously implicated as a risk factor for substance dependence and an objective electroencephalographic feature previously associated with GABRA2 and relapse risk. The second analysis confirmed that the correlation between BMI and the substance-dependence-associated GABRA2 genotype was reliable and sex-specific. Conclusions We conclude that an elevated BMI is associated with genetic, neurophysiological, psychiatric, and psychological indicators of impulsivity. The sex difference may be explained by greater opportunities to eat and overeat, a preference for higher calorie foods, a longer duration of alcohol/drug abstinence, or previous pregnancies in women. PMID:22882390

  13. Phenotypic and genotypic characterization of Streptococcus pneumoniae resistant to macrolide in Casablanca, Morocco.

    PubMed

    Diawara, Idrissa; Zerouali, Khalid; Katfy, Khalid; Barguigua, Abouddihaj; Belabbes, Houria; Timinouni, Mohammed; Elmdaghri, Naima

    2016-06-01

    In Morocco, the 13-valent pneumococcal conjugate vaccine (PCV-13) was introduced in the national immunization program (NIP) in October 2010 and replaced by the PCV-10 in July 2012. The present study aimed to determine the prevalence of erythromycin-resistant Streptococcus pneumoniae (ERSP) and to analyze the phenotypic and genotypic characteristics of these isolates in Casablanca, Morocco from January 2007 to December 2014. Isolates were obtained from the Microbiology Laboratory of Ibn Rochd University Hospital Centre of Casablanca. Serogrouping was done using Pneumotest Kit and serotyping by the Quellung capsular swelling. Antibiotic susceptibility pattern was determined by disk diffusion and Etest methods. A total of 655S. pneumoniae isolates were collected from 2007 to 2014 from pediatric and adult patients. Fifty-five percent of these isolates were from invasive pneumococcal diseases. Of the 655 isolates, 92 (14%) were ERSP. Globally, the proportion of ERSP from 2007 to 2010 (before vaccination) and from 2011 to 2014 (after vaccination) were 11.6% and 17.2% (p=0.04), respectively. Of the 92 ERSP, 89%, 4% and 7% displayed constitutive MLSB (resistance to macrolide, lincosamide and streptogramin B), inducible MLSB, and M phenotype (resistance to macrolide only), respectively. ERSP genotypic analysis showed that 90.2% carried the ermB gene, 6.5% the mefE gene, and 3.3% both the genes (ermB+mefE). The most prevalent ERSP serotypes were 6B, 19F and 23F before vaccination and 19F, 6B, 6A and 23F after vaccination. Erythromycin resistance among S. pneumoniae is relatively high in Casablanca. The contribution of PCVs to the reduction in antibiotic use is encouraging but this should be accompanied by a rational use of antibiotic. PMID:26961592

  14. A fast and low-cost genotyping method for hepatitis B virus based on pattern recognition in point-of-care settings

    PubMed Central

    Qiu, Xianbo; Song, Liuwei; Yang, Shuo; Guo, Meng; Yuan, Quan; Ge, Shengxiang; Min, Xiaoping; Xia, Ningshao

    2016-01-01

    A fast and low-cost method for HBV genotyping especially for genotypes A, B, C and D was developed and tested. A classifier was used to detect and analyze a one-step immunoassay lateral flow strip functionalized with genotype-specific monoclonal antibodies (mAbs) on multiple capture lines in the form of pattern recognition for point-of-care (POC) diagnostics. The fluorescent signals from the capture lines and the background of the strip were collected via multiple optical channels in parallel. A digital HBV genotyping model, whose inputs are the fluorescent signals and outputs are a group of genotype-specific digital binary codes (0/1), was developed based on the HBV genotyping strategy. Meanwhile, a companion decoding table was established to cover all possible pairing cases between the states of a group of genotype-specific digital binary codes and the HBV genotyping results. A logical analyzing module was constructed to process the detected signals in parallel without program control, and its outputs were used to drive a set of LED indicators, which determine the HBV genotype. Comparing to the nucleic acid analysis to HBV viruses, much faster HBV genotyping with significantly lower cost can be obtained with the developed method. PMID:27306485

  15. Identification of a natural intergenotypic recombinant hepatitis delta virus genotype 1 and 2 in Vietnamese HBsAg-positive patients.

    PubMed

    Sy, B T; Nguyen, H M; Toan, N L; Song, L H; Tong, H V; Wolboldt, C; Binh, V Q; Kremsner, P G; Velavan, T P; Bock, C-T

    2015-01-01

    Hepatitis D virus (HDV) infection is acquired as a co- /superinfection of Hepatitis B virus (HBV) and can modulate the pathophysiology of chronic hepatitis B and related liver diseases including hepatocellular carcinoma. Among the eight distinct HDV genotypes reported, relatively few studies have attempted to investigate the prevalence of HDV mixed genotypes and RNA recombination of HDV. With a recorded prevalence of 10-20% HBV infection in Vietnam, this study investigated the HDV variability, HDV genotypes and HDV recombination among twenty-one HDV isolates in Vietnamese HBsAg-positive patients. HDV subgenomic and full-length genome sequences were obtained using newly established HDV-specific RT-PCR techniques. The nucleotide homology was observed from 74.6% to 99.4% among the investigated full-length genome of the HDV isolates. We observed HDV genotype 1 and HDV genotype 2 in the investigated Vietnamese patients. Although no HDV genotype mixtures were observed, we report here a newly identified recombinant of HDV genotypes (HDV 1 and HDV 2). The identified recombinant HDV isolate C03 revealed sequence homology to both HDV genotype 1 (nt1 to nt907) and HDV genotype 2 (nt908 to nt1675; HDAg coding region) with a breakpoint at nt908. Our findings demonstrate the prevalence of intergenotypic recombination between HDV genotypes 1 and 2 in a Vietnamese HBsAg-positive patient. Extended investigation on the distribution and prevalence of HDV, HDV mixed genotypes and recombinant HDV genotypes in a larger Vietnamese population offers vital insights into understanding of the micro-epidemiology of HDV and subsequent pathophysiology in chronic HBV- /HDV-related liver diseases. PMID:24548489

  16. 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. The tMRCA analysis suggests a simultaneous circulation of previously described sub-genotypes rather than recent introductions of the Panamanian sub-genotypes in the country. Moreover, these results highlight the need of more intensive research of the HBV strains circulating in the region at the molecular level. In conclusion, Panama has a high HBV genotype diversity that includes a new proposed sub-genotype, an elevated number of PreCore-Core mutations, and confinement of these variants in a specific geographical location. PMID:25093674

  17. High diversity of hepatitis B virus genotypes in Panamanian blood donors: a molecular analysis of new variants.

    PubMed

    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. The tMRCA analysis suggests a simultaneous circulation of previously described sub-genotypes rather than recent introductions of the Panamanian sub-genotypes in the country. Moreover, these results highlight the need of more intensive research of the HBV strains circulating in the region at the molecular level. In conclusion, Panama has a high HBV genotype diversity that includes a new proposed sub-genotype, an elevated number of PreCore-Core mutations, and confinement of these variants in a specific geographical location. PMID:25093674

  18. Hepatitis B Virus Genotypes Distribution with HBsAg Positive in the North of Iran (Mazandaran) During 2011-2014

    PubMed Central

    Mousavi, Tahoora; Haghshenas, Mohammad Reza; Rafiei, Alireza; Navaei, Reza Alizadeh; khah, Zahra Hosseini

    2014-01-01

    Background: HBV infection is a major global health problem and ten genotypes (A to J) and multiples subtypes of HBV have been identified, and they show some distinct geographic distributions. The available data on HBV genotype in Iran are very heterogeneous and limited. Therefore in this study, we tried to identify the HBV genotypes by using polymerase chain reaction. Methods: In this cross-sectional study, HBV- positive serum samples of 122 patients with chronic hepatitis from 2011 to 2014 were studied. HBV-DNA was extracted from plasma samples using QIAamp® MiniElute® Virus Spin Kit (Qiagen). Plasma samples from HBsAg positive were confirmed the presence of HBV nucleic acid and determined the genotypes of HBV genome by PCR using the DNA PCR kit (Cinagene) with Taq-DNA polymerase enzyme and type of specific primers. All samples were examined in the virology laboratory of Sari Medical School. Results: The mean age of patients were 45 ± 25 (range, 20 to 70) year that 70 (57.37%) patients were male and 52 (42.62%) were female. The majority of HBV positive patients had a major surgery (44% patients) and then 32% patients followed by intra familial of hepatitis B virus infected and 11% of HBV positive patients had a history of blood transfusions. In this study, 91(74.59%) had genotype D, 7(5.73%) genotype B and 24(19.67%) genotype D and B. Conclusion: This study indicates that the genotype D is the most frequent followed by the mixed genotypes D and B and genotype B in our region. Prevalence and incidence of HBV genotypes are with distributed among of areas and different genotypes may show different responses with antiviral therapy. PMID:25648402

  19. 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

  20. Temporal Dynamics and Decay of Putatively Allochthonous and Autochthonous Viral Genotypes in Contrasting Freshwater Lakes

    PubMed Central

    Barbosa, Jorge G.; Brown, Julia M.; Donelan, Ryan P.; Eaglesham, James B.; Eggleston, Erin M.; LaBarre, Brenna A.

    2012-01-01

    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

  1. Microcystis genotype succession and related environmental factors in Lake Taihu during cyanobacterial blooms.

    PubMed

    Wang, Xingyu; Sun, Mengjia; Wang, Jinmei; Yang, Letian; Luo, Lan; Li, Pengfu; Kong, Fanxiang

    2012-11-01

    From spring to autumn, heavy Microcystis blooms always occur in Lake Taihu, although environmental conditions vary markedly. We speculated that Microcystis genotype succession could play an important role in adaptation to environmental changes and long-term maintenance of the high Microcystis biomass. In this study, we investigated Microcystis genotype succession pattern and the related environmental variables in Lake Taihu during cyanobacterial blooms. Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction -amplified the genus-specific cpcBA and mcyJ gene fragments was used to monitor the variations of Microcystis genotype and potential microcystin (MC)-producing Microcystis genotype compositions during blooms biweekly in three sites (Meiliang Bay, lake center, and Gonghu Bay) and CANOCO 4.5 for Windows were used for the multivariate statistical analysis of their relationships to environmental variables. DGGE patterns indicated that the number of dominant cpcBA genotype per sample increased from spring to autumn. Principal component analysis ordination plots of DGGE profiles showed clear temporal distribution pattern, but not spatial distribution pattern based on both cpcBA and mcyJ genotype compositions. These results indicated there were relatively gradual successions of Microcystis cpcBA and mcyJ genotype compositions in each site, and no distinct spatial difference among the three sites. Redundancy analyses of the gel patterns showed that, in all the three sites, three environmental factors (nitrate, pH, and chemical oxygen demand) were correlated significantly to successions of both cpcBA and mcyJ genotypes except for mcyJ genotype in the lake center. Spearman's correlations indicated that the three environmental variables were also strongly correlated with chl a and MC concentrations. These results suggested that the environmental factors affecting succession of Microcystis community composition might also influence the growth of

  2. 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.

  3. Hepatitis B virus genotypes circulating in Brazil: molecular characterization of genotype F isolates

    PubMed Central

    Mello, Francisco CA; Souto, Francisco JD; Nabuco, Leticia C; Villela-Nogueira, Cristiane A; Coelho, Henrique Sergio M; Franz, Helena Cristina F; Saraiva, Joao Carlos P; Virgolino, Helaine A; Motta-Castro, Ana Rita C; Melo, Mabel MM; Martins, Regina MB; Gomes, Selma A

    2007-01-01

    Background Hepatitis B virus (HBV) isolates have been classified in eight genotypes, A to H, which exhibit distinct geographical distributions. Genotypes A, D and F are predominant in Brazil, a country formed by a miscegenated population, where the proportion of individuals from Caucasian, Amerindian and African origins varies by region. Genotype F, which is the most divergent, is considered indigenous to the Americas. A systematic molecular characterization of HBV isolates from different parts of the world would be invaluable in establishing HBV evolutionary origins and dispersion patterns. A large-scale study is needed to map the region-by-region distribution of the HBV genotypes in Brazil. Results Genotyping by PCR-RFLP of 303 HBV isolates from HBsAg-positive blood donors showed that at least two of the three genotypes, A, D, and F, co-circulate in each of the five geographic regions of Brazil. No other genotypes were identified. Overall, genotype A was most prevalent (48.5%), and most of these isolates were classified as subgenotype A1 (138/153; 90.2%). Genotype D was the most common genotype in the South (84.2%) and Central (47.6%) regions. The prevalence of genotype F was low (13%) countrywide. Nucleotide sequencing of the S gene and a phylogenetic analysis of 32 HBV genotype F isolates showed that a great majority (28/32; 87.5%) belonged to subgenotype F2, cluster II. The deduced serotype of 31 of 32 F isolates was adw4. The remaining isolate showed a leucine-to-isoleucine substitution at position 127. Conclusion The presence of genotypes A, D and F, and the absence of other genotypes in a large cohort of HBV infected individuals may reflect the ethnic origins of the Brazilian population. The high prevalence of isolates from subgenotype A1 (of African origin) indicates that the African influx during the colonial slavery period had a major impact on the circulation of HBV genotype A currently found in Brazil. Although most genotype F isolates belonged to

  4. Genotype imputation reference panel selection using maximal phylogenetic diversity.

    PubMed

    Zhang, Peng; Zhan, Xiaowei; Rosenberg, Noah A; Zöllner, Sebastian

    2013-10-01

    The recent dramatic cost reduction of next-generation sequencing technology enables investigators to assess most variants in the human genome to identify risk variants for complex diseases. However, sequencing large samples remains very expensive. For a study sample with existing genotype data, such as array data from genome-wide association studies, a cost-effective approach is to sequence a subset of the study sample and then to impute the rest of the study sample, using the sequenced subset as a reference panel. The use of such an internal reference panel identifies population-specific variants and avoids the problem of a substantial mismatch in ancestry background between the study population and the reference population. To efficiently select an internal panel, we introduce an idea of phylogenetic diversity from mathematical phylogenetics and comparative genomics. We propose the "most diverse reference panel", defined as the subset with the maximal "phylogenetic diversity", thereby incorporating individuals that span a diverse range of genotypes within the sample. Using data both from simulations and from the 1000 Genomes Project, we show that the most diverse reference panel can substantially improve the imputation accuracy compared to randomly selected reference panels, especially for the imputation of rare variants. The improvement in imputation accuracy holds across different marker densities, reference panel sizes, and lengths for the imputed segments. We thus propose a novel strategy for planning sequencing studies on samples with existing genotype data. PMID:23934887

  5. Next-Generation Sequencing-Based HPV Genotyping Assay Validated in Formalin-Fixed, Paraffin-Embedded Oropharyngeal and Cervical Cancer Specimens

    PubMed Central

    Ambulos, Nicholas P.; Schumaker, Lisa M.; Mathias, Trevor J.; White, Ruth; Troyer, Jennifer; Wells, David

    2016-01-01

    Available clinical human papilloma virus (HPV) diagnostics for head and neck cancer have limited sensitivity and/or fail to define the HPV genotype. Common HPV genotyping assays are costly and labor intensive. We sought to develop a next-generation sequencing (NGS)-based HPV genotyping assay that was sensitive enough to work on formalin-fixed paraffin-embedded (FFPE) samples. We developed an ion torrent NGS HPV genotyping assay using barcoded HPV PCR broad-spectrum general primers 5+/6+ (BSGP)5+/6+. To validate genotype specificity and use in archived clinical FFPE tumor samples, we compared NGS HPV genotyping at 2 sequencing centers with typing by Roche Linear Array assay in 42 oropharyngeal and cervical cancer specimens representing 10 HPV genotypes, as well as HPV-negative cases. To demonstrate the detection of a broad range of HPV genotypes, we genotyped a cohort of 266 cervical cancers. A comparison of NGS genotyping of FFPE cancer specimens with genotyping by Linear Array showed concordant results in 34/37 samples (92%) at sequencing site 1 and 39/42 samples (93%) at sequencing site 2. Concordance between sites was 92%. Designed for use with 10 ng genomic DNA, the assay detected HPV using as little as 1.25 ng FFPE-derived genomic DNA. In 266 cervical cancer specimens, the NGS assay identified 20 different HPV genotypes, including all 13 carcinogenic genotypes. This novel NGS assay provides a sensitive and specific high-throughput method to detect and genotype HPV in a range of clinical specimens derived from FFPE with low per-sample cost. PMID:27006646

  6. Next-Generation Sequencing-Based HPV Genotyping Assay Validated in Formalin-Fixed, Paraffin-Embedded Oropharyngeal and Cervical Cancer Specimens.

    PubMed

    Ambulos, Nicholas P; Schumaker, Lisa M; Mathias, Trevor J; White, Ruth; Troyer, Jennifer; Wells, David; Cullen, Kevin J

    2016-07-01

    Available clinical human papilloma virus (HPV) diagnostics for head and neck cancer have limited sensitivity and/or fail to define the HPV genotype. Common HPV genotyping assays are costly and labor intensive. We sought to develop a next-generation sequencing (NGS)-based HPV genotyping assay that was sensitive enough to work on formalin-fixed paraffin-embedded (FFPE) samples. We developed an ion torrent NGS HPV genotyping assay using barcoded HPV PCR broad-spectrum general primers 5(+)/6(+) (BSGP)5(+)/6(+). To validate genotype specificity and use in archived clinical FFPE tumor samples, we compared NGS HPV genotyping at 2 sequencing centers with typing by Roche Linear Array assay in 42 oropharyngeal and cervical cancer specimens representing 10 HPV genotypes, as well as HPV-negative cases. To demonstrate the detection of a broad range of HPV genotypes, we genotyped a cohort of 266 cervical cancers. A comparison of NGS genotyping of FFPE cancer specimens with genotyping by Linear Array showed concordant results in 34/37 samples (92%) at sequencing site 1 and 39/42 samples (93%) at sequencing site 2. Concordance between sites was 92%. Designed for use with 10 ng genomic DNA, the assay detected HPV using as little as 1.25 ng FFPE-derived genomic DNA. In 266 cervical cancer specimens, the NGS assay identified 20 different HPV genotypes, including all 13 carcinogenic genotypes. This novel NGS assay provides a sensitive and specific high-throughput method to detect and genotype HPV in a range of clinical specimens derived from FFPE with low per-sample cost. PMID:27006646

  7. Use of immuno-dominant epitope derived from genotype 4 as a diagnostic reagent for detecting the antibodies against Hepatitis E Virus

    PubMed Central

    2013-01-01

    Background Despite the genotype 4 has become the dominant cause of hepatitis E disease in China, none antigen derived from genotype 4 of hepatitis E virus (HEV) was used in current commercial anti-HEV immunoassay, and the serological reactivity of antigen derive from genotype 4 is not well-charactered. Methods We expressed and purified the 4 main immuno-dominant epitopes derived from genotype 1 and 4 including ORF2 (410-621aa) of genotype 4, ORF3 (47-114aa) of genotype 4, ORF2 (396-606aa) of genotype 1 and ORF3 (56-123aa) of genotype 4. Results The ORF2 of genotype 4 displayed good diagnostics performance according to ROC analysis using in-house panel, and the immunoassays based the ORF2 of genotype 4 was then developed to detect the anti-HEV IgG antibodies and evaluated further in 530 anti-HEV IgG positive specimens and 380 negative specimens. The sensitivity and the specificity is 98.1% (520/530) and 94.7% (360/380) for immunoassay based on ORF2 of genotype 4, 96.6% (512/530) and 92.6% (352/380) for commercial immunoassay based on genotype 1. It is noted that all of the positive samples will be detected by combing two assays together. The anti-HEV immunoassays based on genotype 4 are in accordance with Chinese anti-HEV national standard,and show an good agreement of 95.8% with commercial assay (kappa=0.913, P=0.014). Conclusions The immunoassay based on ORF2G4 displays good performance, and combining assay based on genotype 1 together with genotype 4 will benefit the HEV diagnosis in large scale samples. PMID:23618011

  8. Hepatitis B virus genotype A: design of reference sequences for sub-genotypes.

    PubMed

    Cai, Qun; Zhu, Huilan; Zhang, Yafei; Li, Xu; Zhang, Zhenhua

    2016-06-01

    Genotype A of hepatitis B virus (HBV/A) is widespread and is currently divided into six sub-genotypes. Suitable reference sequences for different sub-genotypes can facilitate research on HBV/A. However, the current reference sequences for this virus are insufficient. In the present work, we retrieved 442 full-length HBV/A genomic sequences from the GenBank database and classified them into sub-genotypes by phylogenetic analysis. By the maximum likelihood method using the MEGA6.0 software, we established the reference sequences for different HBV/A sub-genotypes. Our analyses demonstrated that these reference sequences clustered phylogenetically with known strains, indicating that the reference sequences we established indeed belonged to the right sub-genotypes. HBV/A subtype sequences were selected by geographic origins and grouped as sub-genotypes including A1-South Africa, A2-Europe, A3-Cameroon, and A5-Haiti. Reference sequences of sub-genotypes A1, A2, A3, and A5 were constructed and deposited into GenBank (KP234050-KP234053). By applying phylogenetic analyses, we further determined the time to most recent common ancestor of HBV/A lineages. In conclusion, these newly established reference sequences can provide suitable reference standards for studies on the molecular biology and virology of HBV genotype A. PMID:27002608

  9. Inferring haplotypes from genotypes on a pedigree with mutations, genotyping errors and missing alleles.

    PubMed

    Wang, Wei-Bung; Jiang, Tao

    2011-04-01

    Inferring the haplotypes of the members of a pedigree from their genotypes has been extensively studied. However, most studies do not consider genotyping errors and de novo mutations. In this paper, we study how to infer haplotypes from genotype data that may contain genotyping errors, de novo mutations, and missing alleles. We assume that there are no recombinants in the genotype data, which is usually true for tightly linked markers. We introduce a combinatorial optimization problem, called haplotype configuration with mutations and errors (HCME), which calls for haplotype configurations consistent with the given genotypes that incur no recombinants and require the minimum number of mutations and errors. HCME is NP-hard. To solve the problem, we propose a heuristic algorithm, the core of which is an integer linear program (ILP) using the system of linear equations over Galois field GF(2). Our algorithm can detect and locate genotyping errors that cannot be detected by simply checking the Mendelian law of inheritance. The algorithm also offers error correction in genotypes/haplotypes rather than just detecting inconsistencies and deleting the involved loci. Our experimental results show that the algorithm can infer haplotypes with a very high accuracy and recover 65%-94% of genotyping errors depending on the pedigree topology. PMID:21523936

  10. Phylogeography of Japanese Encephalitis Virus: Genotype Is Associated with Climate

    PubMed Central

    Schuh, Amy J.; Ward, Melissa J.; Leigh Brown, Andrew J.; Barrett, Alan D. T.

    2013-01-01

    The circulation of vector-borne zoonotic viruses is largely determined by the overlap in the geographical distributions of virus-competent vectors and reservoir hosts. What is less clear are the factors influencing the distribution of virus-specific lineages. Japanese encephalitis virus (JEV) is the most important etiologic agent of epidemic encephalitis worldwide, and is primarily maintained between vertebrate reservoir hosts (avian and swine) and culicine mosquitoes. There are five genotypes of JEV: GI-V. In recent years, GI has displaced GIII as the dominant JEV genotype and GV has re-emerged after almost 60 years of undetected virus circulation. JEV is found throughout most of Asia, extending from maritime Siberia in the north to Australia in the south, and as far as Pakistan to the west and Saipan to the east. Transmission of JEV in temperate zones is epidemic with the majority of cases occurring in summer months, while transmission in tropical zones is endemic and occurs year-round at lower rates. To test the hypothesis that viruses circulating in these two geographical zones are genetically distinct, we applied Bayesian phylogeographic, categorical data analysis and phylogeny-trait association test techniques to the largest JEV dataset compiled to date, representing the envelope (E) gene of 487 isolates collected from 12 countries over 75 years. We demonstrated that GIII and the recently emerged GI-b are temperate genotypes likely maintained year-round in northern latitudes, while GI-a and GII are tropical genotypes likely maintained primarily through mosquito-avian and mosquito-swine transmission cycles. This study represents a new paradigm directly linking viral molecular evolution and climate. PMID:24009790

  11. Predictive Value of HIV-1 Genotypic Resistance Test Interpretation Algorithms

    PubMed Central

    Rhee, Soo-Yon; Fessel, W. Jeffrey; Liu, Tommy F.; Marlowe, Natalia M.; Rowland, Charles M.; Rode, Richard A.; Vandamme, Anne-Mieke; Laethem, Kristel Van; Brun-Vezinet, Francçoise; Calvez, Vincent; Taylor, Jonathan; Hurley, Leo; Horberg, Michael; Shafer, Robert W.

    2016-01-01

    Background Interpreting human immunodeficienc virus type 1 (HIV-1) genotypic drug-resistance test results is challenging for clinicians treating HIV-1–infected patients. Multiple drug-resistance interpretation algorithms have been developed, but their predictive value has rarely been evaluated using contemporary clinical data sets. Methods We examined the predictive value of 4 algorithms at predicting virologic response (VR) during 734 treatment-change episodes (TCEs). VR was define as attaining plasma HIV-1 RNA levels below the limit of quantification Drug-specifi genotypic susceptibility scores (GSSs) were calculated by applying each algorithm to the baseline genotype. Weighted GSSs were calculated by multiplying drug-specifi GSSs by antiretroviral (ARV) potency factors. Regimen-specifi GSSs (rGSSs) were calculated by adding unweighted or weighted drug-specif c GSSs for each salvage therapy ARV. The predictive value of rGSSs were estimated by use of multivariate logistic regression. Results Of 734 TCEs, 475 (65%) were associated with VR. The rGSSs for the 4 algorithms were the variables most strongly predictive of VR. The adjusted rGSS odds ratios ranged from 1.6 to 2.2 (P < .001). Using 10-fold cross-validation, the averaged area under the receiver operating characteristic curve for all algorithms increased from 0.76 with unweighted rGSSs to 0.80 with weighted rGSSs. Conclusions Unweighted and weighted rGSSs of 4 genotypic resistance algorithms were the strongest independent predictors of VR. Optimizing ARV weighting may further improve VR predictions. PMID:19552527

  12. A latent modeling approach to genotype-phenotype relationships: maternal problem behavior clusters, prenatal smoking, and MAOA genotype.

    PubMed

    McGrath, L M; Mustanski, B; Metzger, A; Pine, D S; Kistner-Griffin, E; Cook, E; Wakschlag, L S

    2012-08-01

    This study illustrates the application of a latent modeling approach to genotype-phenotype relationships and gene × environment interactions, using a novel, multidimensional model of adult female problem behavior, including maternal prenatal smoking. The gene of interest is the monoamine oxidase A (MAOA) gene which has been well studied in relation to antisocial behavior. Participants were adult women (N = 192) who were sampled from a prospective pregnancy cohort of non-Hispanic, white individuals recruited from a neighborhood health clinic. Structural equation modeling was used to model a female problem behavior phenotype, which included conduct problems, substance use, impulsive-sensation seeking, interpersonal aggression, and prenatal smoking. All of the female problem behavior dimensions clustered together strongly, with the exception of prenatal smoking. A main effect of MAOA genotype and a MAOA × physical maltreatment interaction were detected with the Conduct Problems factor. Our phenotypic model showed that prenatal smoking is not simply a marker of other maternal problem behaviors. The risk variant in the MAOA main effect and interaction analyses was the high activity MAOA genotype, which is discrepant from consensus findings in male samples. This result contributes to an emerging literature on sex-specific interaction effects for MAOA. PMID:22610759

  13. Allelic frequency and genotypes of prion protein at codon 136 and 171 in Iranian Ghezel sheep breeds

    PubMed Central

    Zadeh, Reza Ashrafi; Omrani, Mir Davood; Ramezani, Fatemeh; Amniattalab, Amir

    2011-01-01

    PrP genotypes at codons 136 and 171 in 120 Iranian Ghezel sheep breeds were studied using allele-specific PCR amplification and compared with the well-known sheep breeds in North America, the United States and Europe. The frequency of V allele and VV genotype at codon 136 of Ghezel sheep breed was significantly lower than AA and AV. At codon 171, the frequency of allele H was significantly lower than Q and R. Despite the similarities of PrP genotypes at codons 136 and 171 between Iranian Ghezel sheep breeds and some of the studied breeds, significant differences were found with others. Planning of effective breeding control and successful eradication of susceptible genotypes in Iranian Ghezel sheep breeds will not be possible unless the susceptibility of various genotypes in Ghezel sheep breeds to natural or experimental scrapie has been elucidated. PMID:21778818

  14. Genotypic variations in the dynamics of metal concentrations in poplar leaves: a field study with a perspective on phytoremediation.

    PubMed

    Pottier, Mathieu; García de la Torre, Vanesa S; Victor, Cindy; David, Laure C; Chalot, Michel; Thomine, Sébastien

    2015-04-01

    Poplar is commonly used for phytoremediation of metal polluted soils. However, the high concentrations of trace elements present in leaves may return to soil upon leaf abscission. To investigate the mechanisms controlling leaf metal content, metal concentrations and expression levels of genes involved in metal transport were monitored at different developmental stages on leaves from different poplar genotypes growing on a contaminated field. Large differences in leaf metal concentrations were observed among genotypes. Whereas Mg was remobilized during senescence, Zn and Cd accumulation continued until leaf abscission in all genotypes. A positive correlation between Natural Resistance Associated Macrophage Protein 1 (NRAMP1) expression levels and Zn bio-concentration factors was observed. Principal component analyses of metal concentrations and gene expression levels clearly discriminated poplar genotypes. This study highlights a general absence of trace element remobilization from poplar leaves despite genotype specificities in the control of leaf metal homeostasis. PMID:25622297

  15. Effect of Genotype and Environment on Salvia miltiorrhiza Roots Using LC/MS-Based Metabolomics.

    PubMed

    Zhao, Qi; Song, Zhenqiao; Fang, Xinsheng; Pan, Yuling; Guo, Linlin; Liu, Tian; Wang, Jianhua

    2016-01-01

    Salvia miltiorrhiza (S. miltiorrhiza) Bunge is broadly used as herbal medicine for the clinical treatments of cardiovascular and cerebrovascular diseases. Despite its commercial and medicinal values, few systematic studies on the metabolome of S. miltiorrhiza roots have been carried out so far. We systematically described the metabolic profiles of S. miltiorrhiza using high pressure liquid chromatography mass spectrometry (LC/MS) in conjunction with multivariate statistical analyses, aimed at monitoring their biological variations of secondary metabolites related to three locations and four S. miltiorrhiza genotypes. A total of 40 bioactive constituents were putatively annotated in S. miltiorrhiza root samples. This study found that both the same S. miltiorrhiza genotype growing at three different locations and four S. miltiorrhiza genotypes growing at the same location had significant metabonomic differences identified by the principal component analysis (PCA) approach. By using orthogonal projection to latent structure with discriminant analysis (OPLS-DA), 16 and 14 secondary metabolites can be used as potential location-specific and genotype-specific markers in S. miltiorrhiza, respectively. The specificity of LC/MS profiles offered a powerful tool to discriminate S. miltiorrhiza samples according to genotypes or locations. PMID:27023512

  16. Association of the vitamin D receptor genotype with bone metastases in breast cancer patients.

    PubMed

    Schöndorf, Thomas; Eisberg, Carsten; Wassmer, Gernot; Warm, Mathias; Becker, Martina; Rein, Daniel T; Göhring, Uwe-Jochen

    2003-01-01

    This study was designed in order to evaluate specific vitamin D receptor (VDR) genotypes as indicators of the likelihood of developing osseous metastases in breast cancer patients. Therefore, we determined polymorphisms of the VDR gene in a study group comprising 183 breast cancer patients. Specific fragments spanning over intron 8 and exon 9 of the VDR gene were amplified by polymerase chain reaction. The fragments were then incubated with each of the specific endonucleases APAI, BSMI or TAQI, respectively. The VDR gene polymorphisms were detected by the presence or absence of the particular restriction site using agarose gel electrophoresis. Statistical analyses revealed a significant correlation between both the VDR gene polymorphisms indicated as AA (absence of the APAI restriction site in both alleles) or TT (absence of the TAQI restriction site in both alleles), respectively, and the occurrence of bone metastases. Patients with the AA genotype have a 1.7-fold increased risk of developing bone metastases, whereas patients with the TT genotype have a 0.5-fold risk. Neither other genotypes nor allelic combinations displayed any further correlation with the clinical stage. The data suggest that the AA genotype of the VDR gene might be useful to identify breast cancer patients with a high probability of forming occult bone metastases who are considered to benefit from an adjuvant bone-protective therapy. PMID:12566913

  17. Male-female genotype interactions maintain variation in traits important for sexual interactions and reproductive isolation.

    PubMed

    Castillo, Dean M; Delph, Lynda F

    2016-07-01

    Prezygotic reproductive isolation can evolve quickly when sexual selection drives divergence in traits important for sexual interactions between populations. It has been hypothesized that standing variation for male/female traits and preferences facilitates this rapid evolution and that variation in these traits is maintained by male-female genotype interactions in which specific female genotypes prefer specific male traits. This hypothesis can also explain patterns of speciation when ecological divergence is lacking, but this remains untested because it requires information about sexual interactions in ancestral lineages. Using a set of ancestral genotypes that previously had been identified as evolving reproductive isolation, we specifically asked whether there is segregating variation in female preference and whether segregating variation in sexual interactions is a product of male-female genotype interactions. Our results provide evidence for segregating variation in female preference and further that male-female genotype interactions are important for maintaining variation that selection can act on and that can lead to reproductive isolation. PMID:27271732

  18. An integrative variant analysis pipeline for accurate genotype/haplotype inference in population NGS data.

    PubMed

    Wang, Yi; Lu, James; Yu, Jin; Gibbs, Richard A; Yu, Fuli

    2013-05-01

    Next-generation sequencing is a powerful approach for discovering genetic variation. Sensitive variant calling and haplotype inference from population sequencing data remain challenging. We describe methods for high-quality discovery, genotyping, and phasing of SNPs for low-coverage (approximately 5×) sequencing of populations, implemented in a pipeline called SNPTools. Our pipeline contains several innovations that specifically address challenges caused by low-coverage population sequencing: (1) effective base depth (EBD), a nonparametric statistic that enables more accurate statistical modeling of sequencing data; (2) variance ratio scoring, a variance-based statistic that discovers polymorphic loci with high sensitivity and specificity; and (3) BAM-specific binomial mixture modeling (BBMM), a clustering algorithm that generates robust genotype likelihoods from heterogeneous sequencing data. Last, we develop an imputation engine that refines raw genotype likelihoods to produce high-quality phased genotypes/haplotypes. Designed for large population studies, SNPTools' input/output (I/O) and storage aware design leads to improved computing performance on large sequencing data sets. We apply SNPTools to the International 1000 Genomes Project (1000G) Phase 1 low-coverage data set and obtain genotyping accuracy comparable to that of SNP microarray. PMID:23296920

  19. Ultrahigh-density linkage map for cultivated cucumber (Cucumis sativus L.) using a single-nucleotide polymorphism genotyping array.

    PubMed

    Rubinstein, Mor; Katzenellenbogen, Mark; Eshed, Ravit; Rozen, Ada; Katzir, Nurit; Colle, Marivi; Yang, Luming; Grumet, Rebecca; Weng, Yiqun; Sherman, Amir; Ophir, Ron

    2015-01-01

    Genotyping arrays are tools for high-throughput genotyping, which is beneficial in constructing saturated genetic maps and therefore high-resolution mapping of complex traits. Since the report of the first cucumber genome draft, genetic maps have been constructed mainly based on simple-sequence repeats (SSRs) or on combinations of SSRs and sequence-related amplified polymorphism (SRAP). In this study, we developed the first cucumber genotyping array consisting of 32,864 single-nucleotide polymorphisms (SNPs). These markers cover the cucumber genome with a median interval of ~2 Kb and have expected genotype calls in parents/F1 hybridizations as a training set. The training set was validated with Fluidigm technology and showed 96% concordance with the genotype calls in the parents/F1 hybridizations. Application of the genotyping array was illustrated by constructing a 598.7 cM genetic map based on a '9930' × 'Gy14' recombinant inbred line (RIL) population comprised of 11,156 SNPs. Marker collinearity between the genetic map and reference genomes of the two parents was estimated at R2 = 0.97. We also used the array-derived genetic map to investigate chromosomal rearrangements, regional recombination rate, and specific regions with segregation distortions. Finally, 82% of the linkage-map bins were polymorphic in other cucumber variants, suggesting that the array can be applied for genotyping in other lines. The genotyping array presented here, together with the genotype calls of the parents/F1 hybridizations as a training set, should be a powerful tool in future studies with high-throughput cucumber genotyping. An ultrahigh-density linkage map constructed by this genotyping array on RIL population may be invaluable for assembly improvement, and for mapping important cucumber QTLs. PMID:25874931

  20. Ultrahigh-Density Linkage Map for Cultivated Cucumber (Cucumis sativus L.) Using a Single-Nucleotide Polymorphism Genotyping Array

    PubMed Central

    Rubinstein, Mor; Katzenellenbogen, Mark; Eshed, Ravit; Rozen, Ada; Katzir, Nurit; Colle, Marivi; Yang, Luming; Grumet, Rebecca; Weng, Yiqun; Sherman, Amir; Ophir, Ron

    2015-01-01

    Genotyping arrays are tools for high-throughput genotyping, which is beneficial in constructing saturated genetic maps and therefore high-resolution mapping of complex traits. Since the report of the first cucumber genome draft, genetic maps have been constructed mainly based on simple-sequence repeats (SSRs) or on combinations of SSRs and sequence-related amplified polymorphism (SRAP). In this study, we developed the first cucumber genotyping array consisting of 32,864 single-nucleotide polymorphisms (SNPs). These markers cover the cucumber genome with a median interval of ~2 Kb and have expected genotype calls in parents/F1 hybridizations as a training set. The training set was validated with Fluidigm technology and showed 96% concordance with the genotype calls in the parents/F1 hybridizations. Application of the genotyping array was illustrated by constructing a 598.7 cM genetic map based on a ‘9930’ × ‘Gy14’ recombinant inbred line (RIL) population comprised of 11,156 SNPs. Marker collinearity between the genetic map and reference genomes of the two parents was estimated at R2 = 0.97. We also used the array-derived genetic map to investigate chromosomal rearrangements, regional recombination rate, and specific regions with segregation distortions. Finally, 82% of the linkage-map bins were polymorphic in other cucumber variants, suggesting that the array can be applied for genotyping in other lines. The genotyping array presented here, together with the genotype calls of the parents/F1 hybridizations as a training set, should be a powerful tool in future studies with high-throughput cucumber genotyping. An ultrahigh-density linkage map constructed by this genotyping array on RIL population may be invaluable for assembly improvement, and for mapping important cucumber QTLs. PMID:25874931

  1. Regulation of tillering in sorghum: genotypic effects

    PubMed Central

    Kim, Hae Koo; Luquet, Delphine; van Oosterom, Erik; Dingkuhn, Michael; Hammer, Graeme

    2010-01-01

    Background and Aims Genotypic variation in tillering can be caused by differences in the carbon supply–demand balance within a plant. The aim of this study was to understand and quantify the effects of genotype on tillering as a consequence of the underlying internal competition for carbohydrates. Methods Five sorghum hybrids, derived from inbred lines with a common genetic background and with similar phenology and plant height but contrasting tillering, were grown in five experiments. The experiments covered a wide range in radiation and temperature conditions, so that number of tillers produced varied significantly. Data on leaf area, tiller number, and biomass accumulation and partitioning were collected at regular intervals. To quantify internal plant competition for carbohydrates, a carbohydrate supply–demand index (S/Dindex) was developed and related to variation in tillering. Key Results The appearance of main shoot leaves and tillers was highly co-ordinated across genotypes. High-tillering hybrids had a greater appearance frequency of early tiller ranks than low-tillering hybrids, and this was associated with narrower and hence smaller main shoot leaves. A generalized S/Dindex of internal plant competition accounted for most of the observed variation in maximum tiller number (Ntiller,max) across genotypes. However, genotypic differences in the relationship between the S/Dindex and Ntiller,max suggested that high-tillering hybrids also had a lower S/D threshold at which tillers appeared, possibly associated with hormonal effects. Conclusions The results support the hypothesis that genotypic differences in tillering were associated with differences in plant carbon S/D balance, associated with differences in leaf size and in the threshold at which tillers grow out. The results provide avenues for phenotyping of mapping populations to identify genomic regions regulating tillering. Incorporating the results in crop growth simulation models could provide

  2. Imputation of ungenotyped parental genotypes in dairy and beef cattle from progeny genotypes.

    PubMed

    Berry, D P; McParland, S; Kearney, J F; Sargolzaei, M; Mullen, M P

    2014-06-01

    The objective of this study was to quantify the accuracy of imputing the genotype of parents using information on the genotype of their progeny and a family-based and population-based imputation algorithm. Two separate data sets were used, one containing both dairy and beef animals (n=3122) with high-density genotypes (735 151 single nucleotide polymorphisms (SNPs)) and the other containing just dairy animals (n=5489) with medium-density genotypes (51 602 SNPs). Imputation accuracy of three different genotype density panels were evaluated representing low (i.e. 6501 SNPs), medium and high density. The full genotypes of sires with genotyped half-sib progeny were masked and subsequently imputed. Genotyped half-sib progeny group sizes were altered from 4 up to 12 and the impact on imputation accuracy was quantified. Up to 157 and 258 sires were used to test the accuracy of imputation in the dairy plus beef data set and the dairy-only data set, respectively. The efficiency and accuracy of imputation was quantified as the proportion of genotypes that could not be imputed, and as both the genotype concordance rate and allele concordance rate. The median proportion of genotypes per animal that could not be imputed in the imputation process decreased as the number of genotyped half-sib progeny increased; values for the medium-density panel ranged from a median of 0.015 with a half-sib progeny group size of 4 to a median of 0.0014 to 0.0015 with a half-sib progeny group size of 8. The accuracy of imputation across different paternal half-sib progeny group sizes was similar in both data sets. Concordance rates increased considerably as the number of genotyped half-sib progeny increased from four (mean animal allele concordance rate of 0.94 in both data sets for the medium-density genotype panel) to five (mean animal allele concordance rate of 0.96 in both data sets for the medium-density genotype panel) after which it was relatively stable up to a half-sib progeny group size

  3. North American tree squirrels and ground squirrels with overlapping ranges host different Cryptosporidium species and genotypes.

    PubMed

    Stenger, Brianna L S; Clark, Mark E; Kváč, Martin; Khan, Eakalak; Giddings, Catherine W; Prediger, Jitka; McEvoy, John M

    2015-12-01

    Wildlife-associated Cryptosporidium are an emerging cause of cryptosporidiosis in humans. The present study was undertaken to determine the extent to which North American tree squirrels and ground squirrels host zoonotic Cryptosporidium species and genotypes. Fragments of the Cryptosporidium 18S rRNA and actin genes were amplified and sequenced from fecal samples obtained from three tree squirrel and three ground squirrel species. In tree squirrels, Cryptosporidium was identified in 40.5% (17/42) of American red squirrels (Tamiasciurus hudsonicus), 40.4% (55/136) of eastern gray squirrels (Sciurus carolinensis), and 28.6% (2/7) of fox squirrels (Sciurus niger). Human-pathogenic Cryptosporidium ubiquitum and Cryptosporidium skunk genotype were the most prevalent species/genotypes in tree squirrels. Because tree squirrels live in close proximity to humans and are frequently infected with potentially zoonotic Cryptosporidium species/genotypes, they may be a significant reservoir of infection in humans. In ground squirrels, Cryptosporidium was detected in 70.2% (33/47) of 13-lined ground squirrels (Ictidomys tridecemlineatus), 35.1% (27/77) of black-tailed prairie dogs (Cynomys ludovicianus), and the only golden-mantled ground squirrel (Callospermophilus lateralis) that was sampled. Cryptosporidium rubeyi and ground squirrel genotypes I, II, and III were identified in isolates from these ground squirrel species. In contrast to the Cryptosporidium infecting tree squirrels, these species/genotypes appear to be specific for ground squirrels and are not associated with human disease. PMID:26437239

  4. HBV endemicity in Mexico is associated with HBV genotypes H and G

    PubMed Central

    Roman, Sonia; Panduro, Arturo

    2013-01-01

    Hepatitis B virus (HBV) genotypes have distinct genetic and geographic diversity and may be associated with specific clinical characteristics, progression, severity of disease and antiviral response. Herein, we provide an updated overview of the endemicity of HBV genotypes H and G in Mexico. HBV genotype H is predominant among the Mexican population, but not in Central America. Its geographic distribution is related to a typical endemicity among the Mexicans which is characterized by a low hepatitis B surface antigen seroprevalence, apparently due to a rapid resolution of the infection, low viral loads and a high prevalence of occult B infection. During chronic infections, genotype H is detected in mixtures with other HBV genotypes and associated with other co-morbidities, such as obesity, alcoholism and co-infection with hepatitis C virus or human immunodeficiency virus. Hepatocellular carcinoma prevalence is low. Thus, antiviral therapy may differ significantly from the standard guidelines established worldwide. The high prevalence of HBV genotype G in the Americas, especially among the Mexican population, raises new questions regarding its geographic origin that will require further investigation. PMID:24023487

  5. Molecular genotyping of Echinococcus granulosus in animal and human isolates from Egypt.

    PubMed

    Aaty, H E Abdel; Abdel-Hameed, D M; Alam-Eldin, Y H; El-Shennawy, S F; Aminou, H A; Makled, S S; Darweesh, S K

    2012-02-01

    Despite, Egypt is started to be considered as an emerging endemic area for cystic echinococcosis (CE), no enough data in the literature about the exact status of the genotype in both animals and humans. Therefore, the present study aims to characterize the underlying genotypes that could be responsible for the transmission cycle and for the growing infectivity. Animal isolates were collected from 47 camels and 6 pigs. Human isolates are 31 CE cases including; 21 of hepatic cases, 5 of pulmonary cases and 5 multiple-organ affection cases. Hot-Start specific PCR followed by DNA sequencing for mitochondrial 12S rRNA gene, revealed G1 genotype in one (3.2%) of 31 human isolate only. G6 genotype was detected in all the 53 (100%) animal isolates and in 30 out of 31 (96.8%) human isolate. The Egyptian G6 strain nucleotide sequence revealed 100% homology with an Argentinean reference strain 99% homology with the Kenyan G6 strain. It was concluded that G6 genotype is the predominant genotype in Egypt. PMID:22062047

  6. Challenges in Determining Genotypes for Pharmacogenetics in Allogeneic Hematopoietic Cell Transplant Recipients.

    PubMed

    Langman, Loralie J; Nesher, Lior; Shah, Dimpy P; Azzi, Jacques M; Shpall, Elizabeth J; Rezvani, Katy; Black, John L; Chemaly, Roy F

    2016-09-01

    As part of a pharmacogenetic study, paired blood and oral fluid samples were tested for the IL28B polymorphism (rs12979860) before and after hematopoietic cell transplantation (HCT) to evaluate changes in the genotype and investigate the utility of genotyping in oral fluid in HCT recipients. In 54 patients with leukemia >18 years of age, samples were collected approximately 7 days before HCT and 60 days after HCT. IL28B polymorphism testing was performed using real-time PCR with allele-specific probes. Twenty-four patients had the same genotype as their donors. In 30 patients, the genotype was different from that of the donor. In the oral fluid samples, 4 retained the recipient's genotype, and 18 had a genotype that matched that of the donor. In the remaining 8 patients, the results could not be characterized and appeared to be a combination of both, suggesting mixed proportions of donor and recipient cells. The assumption was that the sloughed epithelial cells of the mouth are of recipient origin. However, oral fluid is a mixture that contains varying numbers of cells of the recipient and immunomodulatory cells from the donor. Therefore, the use of oral fluid after HCT for clinical pharmacogenetics purposes needs further investigation. PMID:27371869

  7. A novel three-round multiplex PCR for SNP genotyping with next generation sequencing.

    PubMed

    Chen, Ke; Zhou, Yu-Xun; Li, Kai; Qi, Li-Xin; Zhang, Qi-Fei; Wang, Mao-Chun; Xiao, Jun-Hua

    2016-06-01

    Owing to the high throughput and low cost, next generation sequencing has attracted much attention for SNP genotyping application for researchers. Here, we introduce a new method based on three-round multiplex PCR to precisely genotype SNPs with next generation sequencing. This method can as much as possible consume the equivalent amount of each pair of specific primers to largely eliminate the amplification discrepancy between different loci. After the PCR amplification, the products can be directly subjected to next generation sequencing platform. We simultaneously amplified 37 SNP loci of 757 samples and sequenced all amplicons on ion torrent PGM platform; 90.5 % of the target SNP loci were accurately genotyped (at least 15×) and 90.4 % amplicons had uniform coverage with a variation less than 50-fold. Ligase detection reaction (LDR) was performed to genotype the 19 SNP loci (as part of the 37 SNP loci) with 91 samples randomly selected from the 757 samples, and 99.5 % genotyping data were consistent with the next generation sequencing results. Our results demonstrate that three-round PCR coupled with next generation sequencing is an efficient and economical genotyping approach. Graphical Abstract The schematic diagram of three-round PCR. PMID:27113460

  8. Rotavirus genotypes in sewage treatment plants and in children hospitalized with acute diarrhea in Italy in 2010 and 2011.

    PubMed

    Ruggeri, Franco M; Bonomo, Paolo; Ianiro, Giovanni; Battistone, Andrea; Delogu, Roberto; Germinario, Cinzia; Chironna, Maria; Triassi, Maria; Campagnuolo, Rosalba; Cicala, Antonella; Giammanco, Giovanni M; Castiglia, Paolo; Serra, Caterina; Gaggioli, Andrea; Fiore, Lucia

    2015-01-01

    Although the molecular surveillance network RotaNet-Italy provides useful nationwide data on rotaviruses causing severe acute gastroenteritis in children in Italy, scarce information is available on rotavirus circulation in the general Italian population, including adults with mild or asymptomatic infection. We investigated the genotypes of rotaviruses present in urban wastewaters and compared them with those of viral strains from clinical pediatric cases. During 2010 and 2011, 285 sewage samples from 4 Italian cities were tested by reverse transcription-PCRs (RT-PCRs) specific for rotavirus VP7 and VP4 genes. Rotavirus was detected in 172 (60.4%) samples, 26 of which contained multiple rotavirus G (VP7 gene) genotypes, for a total of 198 G types. Thirty-two samples also contained multiple P (VP4 gene) genotypes, yielding 204 P types in 172 samples. Genotype G1 accounted for 65.6% of rotaviruses typed, followed by genotypes G2 (20.2%), G9 (7.6%), G4 (4.6%), G6 (1.0%), G3 (0.5%), and G26 (0.5%). VP4 genotype P[8] accounted for 75.0% of strains, genotype P[4] accounted for 23.0% of strains, and the uncommon genotypes P[6], P[9], P[14], and P[19] accounted for 2.0% of strains altogether. These rotavirus genotypes were also found in pediatric patients hospitalized in the same areas and years but in different proportions. Specifically, genotypes G2, G9, and P[4] were more prevalent in sewage samples than among samples from patients, which suggests either a larger circulation of the latter strains through the general population not requiring medical care or their greater survival in wastewaters. A high level of nucleotide identity in the G1, G2, and G6 VP7 sequences was observed between strains from the environment and those from patients. PMID:25344240

  9. Rotavirus Genotypes in Sewage Treatment Plants and in Children Hospitalized with Acute Diarrhea in Italy in 2010 and 2011

    PubMed Central

    Ruggeri, Franco M.; Bonomo, Paolo; Ianiro, Giovanni; Battistone, Andrea; Delogu, Roberto; Germinario, Cinzia; Chironna, Maria; Triassi, Maria; Campagnuolo, Rosalba; Cicala, Antonella; Giammanco, Giovanni M.; Castiglia, Paolo; Serra, Caterina; Gaggioli, Andrea

    2014-01-01

    Although the molecular surveillance network RotaNet-Italy provides useful nationwide data on rotaviruses causing severe acute gastroenteritis in children in Italy, scarce information is available on rotavirus circulation in the general Italian population, including adults with mild or asymptomatic infection. We investigated the genotypes of rotaviruses present in urban wastewaters and compared them with those of viral strains from clinical pediatric cases. During 2010 and 2011, 285 sewage samples from 4 Italian cities were tested by reverse transcription-PCRs (RT-PCRs) specific for rotavirus VP7 and VP4 genes. Rotavirus was detected in 172 (60.4%) samples, 26 of which contained multiple rotavirus G (VP7 gene) genotypes, for a total of 198 G types. Thirty-two samples also contained multiple P (VP4 gene) genotypes, yielding 204 P types in 172 samples. Genotype G1 accounted for 65.6% of rotaviruses typed, followed by genotypes G2 (20.2%), G9 (7.6%), G4 (4.6%), G6 (1.0%), G3 (0.5%), and G26 (0.5%). VP4 genotype P[8] accounted for 75.0% of strains, genotype P[4] accounted for 23.0% of strains, and the uncommon genotypes P[6], P[9], P[14], and P[19] accounted for 2.0% of strains altogether. These rotavirus genotypes were also found in pediatric patients hospitalized in the same areas and years but in different proportions. Specifically, genotypes G2, G9, and P[4] were more prevalent in sewage samples than among samples from patients, which suggests either a larger circulation of the latter strains through the general population not requiring medical care or their greater survival in wastewaters. A high level of nucleotide identity in the G1, G2, and G6 VP7 sequences was observed between strains from the environment and those from patients. PMID:25344240

  10. Association of FMR1 Genotypes with In Vitro Fertilization (IVF) Outcomes Based on Ethnicity/Race

    PubMed Central

    Gleicher, Norbert; Weghofer, Andrea; Lee, Irene H.; Barad, David H.

    2011-01-01

    The FMR1 gene, mapping to an area of the X chromosome closely associated with autoimmunity also affects ovarian reserve, with specific genotypes associated with distinct ovarian aging patterns. They, therefore, could also be associated with differences of in vitro fertilization (IVF) outcomes, reported between races/ethnicities. We analyzed 339 consecutive IVF patients, 232 Caucasian, 59 African and 48 Asian, for FMR1 genotypes, and tested by multiple logistic regressions for associations between race/ethnicity, FMR1 genotype, autoimmunity and pregnancy chances with IVF. FMR1 genotypes were predictive of pregnancy (P = 0.046), het-norm/low most significantly and with decreasing chance in comparison to norm genotypes (OR 0.44; 95% CI 0.23–0.85; P = 0.014). Race/ethnicity was, overall, independently associated (P = 0.03), African demonstrating decreased odds in comparison to Caucasian (OR 0.33. 95%CI 0.13–0.79; P = 0.014). Autoimmunity did not differ but interaction of autoimmunity with FMR1 genotype almost reached significance (P = 0.07). Logistic regression with race/ethnicity and interaction between FMR1 genotype and autoimmunity in the model, demonstrated 2.5-times the odds of being associated with autoimmune positivity (OR 2.5, 1.34–4.55; P = 0.004). FMR1 genotypes offer a possible explanation for differences in IVF outcomes between races/ethnicities. PMID:21526209

  11. Association between TCF7L2 Genotype and Glycemic Control in Diabetic Patients Treated with Gliclazide.

    PubMed

    Javorský, Martin; Babjaková, Eva; Klimčáková, Lucia; Schroner, Zbynek; Zidzik, Jozef; Stolfová, Mária; Salagovič, Ján; Tkáč, Ivan

    2013-01-01

    Previous studies showed associations between variants in TCF7L2 gene and the therapeutic response to sulfonylureas. All sulfonylureas stimulate insulin secretion by the closure of ATP-sensitive potassium (KATP) channel. The aim of the present study was to compare TCF7L2 genotype specific effect of gliclazide binding to KATP channel A-site (Group 1) with sulfonylureas binding to AB-site (Group 2). A total of 101 patients were treated with sulfonylureas for 6 months as an add-on therapy to the previous metformin treatment. TCF7L2 rs7903146 C/T genotype was identified by real-time PCR with subsequent melting curve analysis. Analyses using the dominant genetic model showed significantly higher effect of gliclazide in the CC genotype group in comparison with combined CT + TT genotype group (1.32 ± 0.15% versus 0.73 ± 0.11%, P (adj) = 0.005). No significant difference in ΔHbA1c between the patients with CC genotype and the T-allele carriers was observed in Group 2. In the multivariate analysis, only the TCF7L2 genotype (P = 0.006) and the baseline HbA1c (P < 0.001) were significant predictors of ΔHbA1c. After introducing an interaction term between the TCF7L2 genotype and the sulfonylurea type into multivariate model, the interaction became a significant predictor (P = 0.023) of ΔHbA1c. The results indicate significantly higher difference in ΔHbA1c among the TCF7L2 genotypes in patients treated with gliclazide than in patients treated with glimepiride, glibenclamide, or glipizide. PMID:23509454

  12. High-throughput RAD-SNP genotyping for characterization of sugar beet genotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High-throughput SNP genotyping provides a rapid way of developing resourceful set of markers for delineating the genetic architecture and for effective species discrimination. In the presented research, we demonstrate a set of 192 SNPs for effective genotyping in sugar beet using high-throughput mar...

  13. Field Response of Sugarcane Genotypes to Freeze Stress with Genotype x Environment Effects on Quality Traits

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Freeze stress is a constraint to sugarcane (Saccharum sp.) with negative effect on sucrose yield, particularly during the harvest season. To understand its impact on the performance of genotypes developed by the Canal Point (CP) breeding program, the genotype by environment (GxE) interaction was app...

  14. Giardia duodenalis genotypes in domestic and wild animals from Romania identified by PCR-RFLP targeting the gdh gene.

    PubMed

    Adriana, Gyӧrke; Zsuzsa, Kalmár; Mirabela Oana, Dumitrache; Mircea, Gherman Călin; Viorica, Mircean

    2016-02-15

    Sixty Giardia duodenalis isolates from domestic (n=49) and wild (n=11) animals (dogs, cats, deers, wolves, raccoon dog and muskrat) were analysed by PCR-RFLP at glutamate dehydrogenase locus (gdh). The isolates were obtained from positive feces samples for Giardia cysts analysed by flotation technique with saturated sodium chloride solution (specific gravity 1.28). Three G. duodenalis genotypes were identified: C (10/60; 16.7%); D (42/60; 70.0%); and E (7/60; 11.7%). In dogs all three genotypes were found, with the following prevalences: 76.9% genotype D (30/39); 23.1% C (9/39); 2.6% genotype E (1/39). One dog was co-infected with C and D genotypes. In cats we identified only G. duodenalis genotype D. Wolves and raccoon dog harbored infection with G. duodenalis genotype D, deers with E type and muskrat C type. This is the first study regarding genotyping of G. duodenalis in cats and wild animals from Romania. To the best of our knowledge, this is the first report of assemblages E in roe deers; assemblage C in wolves and muskrat; and assemblage D in raccoon dog. PMID:26827864

  15. Prevalence and Genotype Distribution of HPV Infection in China: Analysis of 51,345 HPV Genotyping Results from China's Largest CAP Certified Laboratory

    PubMed Central

    Zeng, Zhengyu; Yang, Huaitao; Li, Zaibo; He, Xuekui; Griffith, Christopher C.; Chen, Xiamen; Guo, Xiaolei; Zheng, Baowen; Wu, Shangwei; Zhao, Chengquan

    2016-01-01

    Introduction: The prevalence of cervical Human Papillomavirus (HPV) infection varies greatly worldwide and data regarding HPV prevalence and genotypes in China are limited. Methods: HPV testing results were retrospectively examined at KingMed Diagnostics, the largest independent pathology laboratory in China, from January 2011 to June 2014. All testing was performed using the 26 HPV Genotyping Panel of TellgenplexTM xMAP™ HPV DNA Test assay (TELLGEN, Shanghai, China). Overall prevalence, age-specific prevalence and genotype distributions were analyzed. Results: A total of 51,345 samples were tested and the overall HPV prevalence was 26%, with 21.12% positive for high risk (HR) HPV and 8.37% positive for low risk HPV. 80% of HPV positive cases were positive for a single HPV type. The three most common HR HPV types detected were HPV-52, -16, and -58, in descending order. HPV-18 was only the 6th most common type. When women were divided into three age groups: <30, 30-49, ≥50 years, HR HPV had the highest prevalence rate in women <30 years, and the lowest rate in women 30-49 years of age. The distribution of HR HPV genotypes also varied among these three age groups. Conclusions: To the best of our knowledge, this is largest routine clinical practice report of HPV prevalence and genotypes in a population of women having limited cervical cancer screening. HPV-52 was the most prevalent HR HPV type in this population of women followed by HPV-16 and HPV-58. The overall and age-specific prevalence and genotype distribution of HR HPV are different in this Chinese population compared to that reported from Western countries. PMID:27326245

  16. Dog's genotype of Giardia duodenalis in human: first evidence in Europe.

    PubMed

    Štrkolcová, Gabriela; Maďar, Marián; Hinney, Barbara; Goldová, Mária; Mojžišová, Jana; Halánová, Monika

    2015-12-01

    The unicellular parasite Giardia duodenalis has been divided to eight assemblages (A-H) from which A and B have the most important zoonotic potential. All remaining genotypes have a strong commitment to various host animals. We present here the first clinical case of a human infection with the dog-specific genotype C of G. duodenalis in Slovakia. The patient, 44-year-old woman, suffered from long-term diarrhoea, abdominal pain, anorexia, weight loss, severe itching and dermatitis in the perianal area. The initial microscopic diagnosis was completed by a nested polymerase chain reaction (PCR) which revealed the first evidence of human giardiasis caused by the dog-specific genotype of G. duodenalis on a European scale. A possible role of dogs in zoonotic transmission of giardiasis and its epidemiological and public health relevance is accentuated. PMID:26408607

  17. Estimation of Genotype Distributions and Posterior Genotype Probabilities for β-Mannosidosis in Salers Cattle

    PubMed Central

    Taylor, J. F.; Abbitt, B.; Walter, J. P.; Davis, S. K.; Jaques, J. T.; Ochoa, R. F.

    1993-01-01

    β-Mannosidosis is a lethal lysosomal storage disease inherited as an autosomal recessive in man, cattle and goats. Laboratory assay data of plasma β-mannosidase activity represent a mixture of homozygous normal and carrier genotype distributions in a proportion determined by genotype frequency. A maximum likelihood approach employing data transformations for each genotype distribution and assuming a diallelic model of inheritance is described. Estimates of the transformation and genotype distribution parameters, gene frequency, genotype fitness and carrier probability were obtained simultaneously from a sample of 2,812 observations on U.S. purebred Salers cattle with enzyme activity, age, gender, month of pregnancy, month of testing, and parents identified. Transformations to normality were not required, estimated gene and carrier genotype frequencies of 0.074 and 0.148 were high, and the estimated relative fitness of heterozygotes was 1.36. The apparent overdominance in fitness may be due to a nonrandom sampling of progeny genotypes within families. The mean of plasma enzyme activity was higher for males than females, higher in winter months, lower in summer months and decreased with increased age. Estimates of carrier probabilities indicate that the test is most effective when animals are sampled as calves, although effectiveness of the plasma assay was less for males than females. Test effectiveness was enhanced through averaging repeated assays of enzyme activity on each animal. Our approach contributes to medical diagnostics in several ways. Rather than assume underlying normality for the distributions comprising the mixture, we estimate transformations to normality for each genotype distribution simultaneously with all other model parameters. This process also excludes potential biases due to data preadjustment for systematic effects. We also provide a method for partitioning phenotypic variation within each genotypic distribution which allows an assessment

  18. FMR1 Genotype with Autoimmunity-Associated Polycystic Ovary-Like Phenotype and Decreased Pregnancy Chance

    PubMed Central

    Gleicher, Norbert; Weghofer, Andrea; Lee, Irene H.; Barad, David H.

    2010-01-01

    The FMR1 gene partially appears to control ovarian reserve, with a specific ovarian sub-genotype statistically associated with a polycystic ovary (PCO)- like phenotype. Some forms of PCO have been associated with autoimmunity. We, therefore, investigated in multiple regression analyses associations of ovary-specific FMR1 genotypes with autoimmunity and pregnancy chances (with in vitro fertilization, IVF) in 339 consecutive infertile women (455 IVF cycles), 75 with PCO-like phenotype, adjusted for age, race/ethnicity, medication dosage and number of oocytes retrieved. Patients included 183 (54.0%) with normal (norm) and 156 (46%) with heterozygous (het) FMR1 genotypes; 133 (39.2%) demonstrated laboratory evidence of autoimmunity: 51.1% of het-norm/low, 38.3% of norm and 24.2% het-norm/high genotype and sub-genotypes demonstrated autoimmunity (p = 0.003). Prevalence of autoimmunity increased further in PCO-like phenotype patients with het-norm/low genotype (83.3%), remained unchanged with norm (34.0%) and decreased in het-norm/high women (10.0%; P<0.0001). Pregnancy rates were significantly higher with norm (38.6%) than het-norm/low (22.2%, p = 0.001). FMR1 sub-genotype het-norm/low is strongly associated with autoimmunity and decreased pregnancy chances in IVF, reaffirming the importance of the distal long arm of the X chromosome (FMR1 maps at Xq27.3) for autoimmunity, ovarian function and, likely, pregnancy chance with IVF. PMID:21179569

  19. Roles of human apolipoprotein E in the infectivity and replication of hepatitis C virus genotype 2a.

    PubMed

    Jung, Bo-Kyoung; Kim, Hye-Ran; Park, Gyu-Nam; Luo, Guangxiang; Chang, Kyung-Soo

    2016-06-01

    Hepatitis C virus (HCV) infection is associated with lipoproteins, and apolipoprotein E (apoE) plays an essential role in infectious HCV particles. Although the role of apoE in HCV infection is well known, its role in the replication of HCV remains unclear. The aims of this study were to determine the role of apoE in the RNA replication of major HCV genotypes 1b and 2a, and to determine whether this role is HCVgenotype-dependent using HCV genotype 1b replicon cells and HCV genotype 2a producing (HP) cells. HCV infection was blocked in Huh7.5 cells treated with low-density lipoproteins, very low-density lipoproteins, or apoE3. An apoE3-specific monoclonal antibody also efficiently neutralized HCV infectivity, and HCV infection was dramatically suppressed by the knockdown of apoE expression with an apoE-specific small interfering RNA, suggesting a requirement for apoE in infectious HCV particles. HCV RNA replication was not affected in HP cells treated with each apoE isoform or transfected with apoE-specific siRNAs. However, the knockdown of apoE expression suppressed RNA replication of HCV genotype 1b. The siRNA-mediated knockdown of apoE, apoA1, and apoB expression also suppressed the RNA replication of HCV genotype 1b, but not that of HCV genotype 2a. Taken together, these findings indicate that apoE plays an important role in HCV genotype 2a infection and in HCV genotype 1b RNA replication, but not in the replication of HCV genotype 2a. These results provide important information for the future development of HCV-genotypespecific anti-HCV agents. PMID:27225463

  20. Clinical significance of human papillomavirus genotyping

    PubMed Central

    Choi, Youn Jin

    2016-01-01

    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. PMID:26768784

  1. Comparison of Hybribio GenoArray and Roche Human Papillomavirus (HPV) Linear Array for HPV Genotyping in Anal Swab Samples

    PubMed Central

    Silver, Michelle I.; Brown, Brandon J.; Leng, Chan Yoon; Blas, Magaly M.; Gravitt, Patti E.; Woo, Yin Ling

    2014-01-01

    Human papillomavirus (HPV) is causally associated with anal cancer, as HPV DNA is detected in up to 90% of anal intraepithelial neoplasias and anal cancers. With the gradual increase of anal cancer rates, there is a growing need to establish reliable and clinically relevant methods to detect anal cancer precursors. In resource-limited settings, HPV DNA detection is a potentially relevant tool for anal cancer screening. Here, we evaluated the performance of the Hybribio GenoArray (GA) for genotyping HPV in anal samples, against the reference standard Roche Linear Array (LA). Anal swab samples were obtained from sexually active men who have sex with men. Following DNA extraction, each sample was genotyped using GA and LA. The overall interassay agreement, type-specific, and single and multiple genotype agreements were evaluated by kappa statistics and McNemar's χ2 tests. Using GA and LA, 68% and 76% of samples were HPV DNA positive, respectively. There was substantial interassay agreements for the detection of all HPV genotypes (κ = 0.70, 86% agreement). Although LA was able to detect more genotypes per sample, the interassay agreement was acceptable (κ = 0.53, 63% agreement). GA had poorer specific detection of HPV genotypes 35, 42, and 51 (κ < 0.60). In conclusion, GA and LA showed good interassay agreement for the detection of most HPV genotypes in anal samples. However, the detection of HPV DNA in up to 76% of anal samples warrants further evaluation of its clinical significance. PMID:25502520

  2. The curli biogenesis genes expression level is unassociated with Enterobacter cloacae hsp60 clusters and PFGE genotypes.

    PubMed

    Akbari, Majid; Bakhshi, Bita; Najar-Peerayeh, Shahin; Behmanesh, Mehrdad

    2016-09-01

    The objective of this study was to determine the correlation between Enterobacter cloacae complex subspecies and clusters involved in UTI infections and specific pulsotypes, and to assess the contribution of major curli biogenesis genes (csgD, csgA) expression level to pathogenesis of clusters and genotypes. Based on the PFGE analysis, 37 different profiles were observed among which 8 profiles were common types. Real time PCR of csgD and csgA genes of 50 E. cloacae complex in relation to PFGE and hsp60 genotypes showed that all the genetic clusters are not equally involved in pathogenesis of urinary tract infections. It was elucidated in this study that isolates with common PFGE genotypes belonged to identical hsp60 clusters, and the foremost clusters (VI, III, and V) mainly comprised within PFGE common types. In our study, no significant correlation was detected between the specific hsp60 clusters or PFGE genotypes and the expression level of csgD and csgA genes (P-value > 0.05). This is the first study describing that unequivalent contribution of E. cloacae genotypes and clusters in pathogenesis of UTI, is not owing to varied curli biogenesis expression potential. The PFGE genotyping showed more discriminatory power than hsp60 genotyping for epidemiological studies and source tracking purpose. PMID:27354208

  3. Characterization of Giardia lamblia genotypes in dogs from Tucson, Arizona using SSU-rRNA and β-giardin sequences.

    PubMed

    Johansen, K M; Castro, N S; Lancaster, K E; Madrid, E; Havas, A; Simms, J; Sterling, C R

    2014-01-01

    The objective of this study was to determine if human genotypes of Giardia lamblia could be found in canine companion animals from urban and peri-urban environments in Tucson, Arizona. Canine fecal samples collected from the Humane Society of Southern Arizona between July 2006 and April 2009 were screened for G. lamblia infection using immunofluorescent microscopy and confirmed by polymerase chain reaction (PCR). Of the 672 samples screened, 196 were found positive by IFA and 185 of those positive were successfully amplified through PCR. Sequencing analysis showed samples were primarily of the C or D genotypes (n =154), or showing a mix of the C and D genotypes (n =10). One sample showed a mixed infection of a human genotype (A) and a dog-specific genotype (C). These data are consistent with previous studies showing dog specific genotypes to be dominant in environments where dog-to-dog transmission is likely to occur, and provides further evidence that multiple genes should be targeted for more accurate genotype characterization. PMID:24233408

  4. Comparison of Hybribio GenoArray and Roche human papillomavirus (HPV) linear array for HPV genotyping in anal swab samples.

    PubMed

    Low, Huey Chi; Silver, Michelle I; Brown, Brandon J; Leng, Chan Yoon; Blas, Magaly M; Gravitt, Patti E; Woo, Yin Ling

    2015-02-01

    Human papillomavirus (HPV) is causally associated with anal cancer, as HPV DNA is detected in up to 90% of anal intraepithelial neoplasias and anal cancers. With the gradual increase of anal cancer rates, there is a growing need to establish reliable and clinically relevant methods to detect anal cancer precursors. In resource-limited settings, HPV DNA detection is a potentially relevant tool for anal cancer screening. Here, we evaluated the performance of the Hybribio GenoArray (GA) for genotyping HPV in anal samples, against the reference standard Roche Linear Array (LA). Anal swab samples were obtained from sexually active men who have sex with men. Following DNA extraction, each sample was genotyped using GA and LA. The overall interassay agreement, type-specific, and single and multiple genotype agreements were evaluated by kappa statistics and McNemar's χ(2) tests. Using GA and LA, 68% and 76% of samples were HPV DNA positive, respectively. There was substantial interassay agreements for the detection of all HPV genotypes (κ = 0.70, 86% agreement). Although LA was able to detect more genotypes per sample, the interassay agreement was acceptable (κ = 0.53, 63% agreement). GA had poorer specific detection of HPV genotypes 35, 42, and 51 (κ < 0.60). In conclusion, GA and LA showed good interassay agreement for the detection of most HPV genotypes in anal samples. However, the detection of HPV DNA in up to 76% of anal samples warrants further evaluation of its clinical significance. PMID:25502520

  5. Performance of a Polymer-Based DNA Chip Platform in Detection and Genotyping of Human Papillomavirus in Clinical Samples▿

    PubMed Central

    Schenk, T.; Brandstetter, T.; zur Hausen, A.; Alt-Mörbe, J.; Huzly, D.; Rühe, J.

    2009-01-01

    Human papillomavirus (HPV) plays a key role in the development of cervical and laryngeal cancers. The aim of our study was to compare the performance of a new hydrogel-based HPV genotyping biochip assay (Biochip) to a commercially available and CE-marked conventional PCR followed by reverse hybridization (GenID-PCR). One hundred twenty-three samples were available for the study. Of these samples, 101/123 were gynecological swabs, 8/123 were swabs or biopsy samples of genital warts, 7/123 were biopsy samples of otorhinolaryngeal lesions, 5/123 were samples of skin warts, and 2/123 were samples of orolabial abnormalities. These molecular methods for HPV genotyping showed comparable sensitivity and specificity. However, 19/123 of the results were discrepant. Specifically, Biochip showed better performance in the detection of multiple infections, especially when more than one high-risk genotype was present. Due to the different probe configurations used in the two assays, GenID-PCR achieves only group-specific detection of many HPV genotypes, whereas Biochip allows for specific identification. Overall, the newly developed HPV chip system (Biochip) proved to be a suitable tool for HPV detection and genotyping; it also proved to be superior for establishing HPV genotyping methods. PMID:19279180

  6. Host genotype by parasite genotype interactions underlying the resistance of anopheline mosquitoes to Plasmodium falciparum

    PubMed Central

    Lambrechts, Louis; Halbert, Jean; Durand, Patrick; Gouagna, Louis C; Koella, Jacob C

    2005-01-01

    Background Most studies on the resistance of mosquitoes to their malaria parasites focus on the response of a mosquito line or colony against a single parasite genotype. In natural situations, however, it may be expected that mosquito-malaria relationships are based, as are many other host-parasite systems, on host genotype by parasite genotype interactions. In such systems, certain hosts are resistant to one subset of the parasite's genotypes, while other hosts are resistant to a different subset. Methods To test for genotype by genotype interactions between malaria parasites and their anopheline vectors, different genetic backgrounds (families consisting of the F1 offspring of individual females) of the major African vector Anopheles gambiae were challenged with several isolates of the human malaria parasite Plasmodium falciparum (obtained from naturally infected children in Kenya). Results Averaged across all parasites, the proportion of infected mosquitoes and the number of oocysts found in their midguts were similar in all mosquito families. Both indices of resistance, however, differed considerably among isolates of the parasite. In particular, no mosquito family was most resistant to all parasites, and no parasite isolate was most infectious to all mosquitoes. Conclusions These results suggest that the level of mosquito resistance depends on the interaction between its own and the parasite's genotype. This finding thus emphasizes the need to take into account the range of genetic diversity exhibited by mosquito and malaria field populations in ideas and studies concerning the control of malaria. PMID:15644136

  7. Genotypic Context and Epistasis in Individuals and Populations.

    PubMed

    Sackton, Timothy B; Hartl, Daniel L

    2016-07-14

    Genes encode components of coevolved and interconnected networks. The effect of genotype on phenotype therefore depends on genotypic context through gene interactions known as epistasis. Epistasis is important in predicting phenotype from genotype for an individual. It is also examined in population studies to identify genetic risk factors in complex traits and to predict evolution under selection. Paradoxically, the effects of genotypic context in individuals and populations are distinct and sometimes contradictory. We argue that predicting genotype from phenotype for individuals based on population studies is difficult and, especially in human genetics, likely to result in underestimating the effects of genotypic context. PMID:27419868

  8. Encephalitozoon cuniculi Genotype I as a Causative Agent of Brain Abscess in an Immunocompetent Patient▿

    PubMed Central

    Ditrich, Oleg; Chrdle, Aleš; Sak, Bohumil; Chmelík, Václav; Kubále, Jiří; Dyková, Iva; Kváč, Martin

    2011-01-01

    A brain abscess caused by Encephalitozoon cuniculi genotype I together with Streptococcus intermedius occurred in a patient without major immunocompromise and with diabetes. The distinguishing clinical signs were hemiparesis and epilepsy. The microsporidium was observed in the abscess aspirate, and its specific DNA was also detected in stool and urine. The patient was successfully treated with albendazole and mebendazole. PMID:21593268

  9. Tuber blight development in potato cultivars in response to different genotypes of Phytophthora infestans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Potato late blight, caused by Phytophthora infestans, is one of the most devastating diseases in potatoes, causing significant loses under disease-conducive conditions. Migrations or introduction of new genotypes to a specific region impose a different set of criteria for consideration for potato gr...

  10. Mutation Scanning and Genotyping in Plants by High Resolution DNA Melting

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High-resolution melting analysis after PCR allows closed-tube mutation scanning and genotyping without processing, labeled probes, real-time monitoring or allele-specific amplification. PCR is performed in the presence of the saturating dye, LCGreen® Plus, with subsequent high-resolution melting ana...

  11. Fluorescent directed heteroduplex analysis enhances PCR-based DQA1 and DQB1 genotyping

    SciTech Connect

    Zimmerman, P.A.; Mansfield, E.S.; Miyasaki, T.

    1994-09-01

    We previously showed how directed heteroduplex analysis (DHDA) simplifies DQA1 and DQB1 genotyping and have used the technique to identify a new DQA1 allele (DQA{sup *}0502, which has a single nucleotide difference from DQA1{sup *}0501). In DHDA, labeled probes are mixed with unlabeled PCR products amplified from patient genomic DNA. After controlled re-annealing, allelic heteroduplexes are resolved on polyacrylamide gels (5%, 2.7 M urea). To utilize fluorescence imaging for detecting the heteroduplexes in HLA-typing, probes are labeled by PCR amplification using locus-specific generic primers and gels scanned using the Fluorimager{trademark} 575 (Molecular Dynamics, Inc.). We generate 2-color DHDA probes using locus-specific PCR primers 5{prime}-end labeled with the fluorochromes FAM (positive-strand primer) and JOE (negative-strand primer) (Perkin-Elmer). Genotypic analysis within families obtained from the CEPH repository have been performed by fluorescence-based DHDA. Results to date show 100% concordance between DHDA and sequence-specific oligonucleotide probe (SSOP) genotyping. Fluorescence-based DHDA is performed with fewer probes than SSOP (1 set of locus-specific probes for DHDA and 10 SSOP probes for DQA1 typing or 13 SSOP probes for DQB1 typing). In addition, fluorescent DHDA allows rapid assessment of genotype, aproximately four hours from receipt of sample to typing result. These results suggest that fluorescent DHDA may facilitate DNA-based HLA-typing within the time constraints required for solid organ transplantation.

  12. Semiquantitative Multiplexed Tandem PCR for Detection and Differentiation of Four Theileria orientalis Genotypes in Cattle

    PubMed Central

    Perera, Piyumali K.; Gasser, Robin B.; Firestone, Simon M.; Smith, Lee; Roeber, Florian

    2014-01-01

    Oriental theileriosis is an emerging, tick-borne disease of bovines in the Asia-Pacific region and is caused by one or more genotypes of the Theileria orientalis complex. This study aimed to establish and validate a multiplexed tandem PCR (MT-PCR) assay using three distinct markers (major piroplasm surface protein, 23-kDa piroplasm membrane protein, and the first internal transcribed spacer of nuclear DNA), for the simultaneous detection and semiquantification of four genotypes (Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex. Analytical specificity, analytical sensitivity, and repeatability of the established MT-PCR assay were assessed in a series of experiments. Subsequently, the assay was evaluated using 200 genomic DNA samples collected from cattle from farms on which oriental theileriosis outbreaks had occurred, and 110 samples from a region where no outbreaks had been reported. The results showed the MT-PCR assay specifically and reproducibly detected the expected genotypes (i.e., genotypes Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex, reliably differentiated them, and was able to detect as little as 1 fg of genomic DNA from each genotype. The diagnostic specificity and sensitivity of the MT-PCR were estimated at 94.0% and 98.8%, respectively. The MT-PCR assay established here is a practical and effective diagnostic tool for the four main genotypes of T. orientalis complex in Australia and should assist studies of the epidemiology and pathophysiology of oriental theileriosis in the Asia-Pacific region. PMID:25339402

  13. A multiplex PCR for the simultaneous detection and genotyping of the Echinococcus granulosus complex.

    PubMed

    Boubaker, Ghalia; Macchiaroli, Natalia; Prada, Laura; Cucher, Marcela A; Rosenzvit, Mara C; Ziadinov, Iskender; Deplazes, Peter; Saarma, Urmas; Babba, Hamouda; Gottstein, Bruno; Spiliotis, Markus

    2013-01-01

    Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1-G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1-G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6-G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus. PMID:23350011

  14. Tradeoff between no-call reduction in genotyping error rate and loss of sample size for genetic case/control association studies.

    PubMed

    Kang, S J; Gordon, D; Brown, A M; Ott, J; Finch, S J

    2004-01-01

    Single nucleotide polymorphisms (SNP) may be genotyped for use in case-control designs to test for association between a SNP marker and a disease using a 2 x 3 chi-squared test of independence. Genotyping is often based on underlying continuous measurements, which are classified into genotypes. A "no-call" procedure is sometimes used in which borderline observations are not classified. This procedure has the simultaneous effect of reducing the genotype error rate and the expected number of genotypes observed. Both quantities affect the power of the statistic. We develop methods for calculating the genotype error rate, the expected number of genotypes observed, and the expected power of the resulting test as a function of the no-call procedure. We examine the statistical properties of the chi-squared test using a no-call procedure when the underlying continuous measure of genotype classification is a three-component mixture of univariate normal distributions under a range of parameter specifications. The genotype error rate decreases as the no-call region is increased. The expected number of observations genotyped also decreases. Our key finding is that the expected power of the chi-squared test is not sensitive to the no-call procedure. That is, the benefits of reduced genotype error rate are almost exactly balanced by the losses due to reduced genotype observations. For an underlying univariate normal mixture of genotype classification to be analyzed with a 2 x 3 chi-squared test, there is little, if any, increase in power using a no-call procedure. PMID:14992497

  15. ApoE (Apolipoprotein E) Genotyping

    MedlinePlus

    ... There are no clear-cut tests to diagnose Alzheimer disease during life. Health practitioners can, however, make a reasonably accurate clinical diagnosis of AD by ruling out other potential causes of dementia and checking for a genetic predisposition to AD with APOE genotyping as supplemental ...

  16. Camelina: Adaptation and performance of genotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Camelina (Camelina sativa L. Crantz) has shown potential as an alternative and biofuel crop in cereal-based cropping systems. Our study investigated the adaption, performance, and yield stability among camelina genotypes across diverse US Pacific Northwest (PNW) environments. Seven named camelina ge...

  17. Selection of Shade Tolerant Tall Fescue Genotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tall fescue (Schedonorus arundinaceus) is genetically variable for many agronomic traits, so it might be possible to increase its persistence and productivity in shaded agroforestry applications. The objective of this research was to identify high yielding, shade-tolerant genotypes. Seed was obtai...

  18. Infectivity of the cervine genotype of Cryptosporidium

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cryptosporidiosis is a diarrheal disease of humans and animals caused by parasites in the genus Cryptosporidium, a genus comprising 19 valid species and 40 genotypes. Most human infections are caused by C. hominis and C. parvum. To a lesser extent infections with C. meleagridis, C. felis, C. canis, ...

  19. Distribution of Human Papillomavirus Genotypes in Iranian Women According to the Severity of the Cervical Lesion

    PubMed Central

    Salehi-Vaziri, Mostafa; Sadeghi, Farzin; Hashemi, Firoozeh Sadat; Haeri, Hayedeh; Bokharaei-Salim, Farah; Monavari, Seyed Hamidreza; Keyvani, Hossein

    2016-01-01

    Background Persistent infection with high-risk human papillomavirus (HPV) has been recognized as a major cause of cervical cancer. Distribution of HPV genotypes may differ according to the geographic region and the severity of the cervical lesion. Determining HPV genotypes’ specific distribution is useful for HPV surveillance and control programs. However, little is known about the distribution of HPV genotypes in Iranian women. Objectives The aim of this study was to determine the distribution of HPV genotypes in Iranian women with different grades of cervical lesions. Patients and Methods From 2011 to 2013, a total of 436 Iranian women with convenience sampling strategy were included in this cross-sectional study. In detail, 287 women negative for intraepithelial lesion or malignancy, 32 with atypical squamous cells of undetermined significance (ASCUS), 50 with low-grade squamous intraepithelial lesion (LSIL), 44 with high-grade squamous intraepithelial lesion (HSIL), and 23 with cervical cancer were evaluated in this investigation. HPV genotypes were determined by INNO-LiPA HPV Genotyping Extra assay. Results In total, HPV infection was detected in 45.4% of the cases. The most common high-risk HPV (HR-HPV) genotype was HPV-16 (32.8%), followed by HPV-53 (9.1%). Within low-risk (LR-HPV) genotypes HPV-6 (22.2%) and HPV-44 (6.1%) were the most prevalent. HPV-16 was the predominant genotype in cases with cervical cancer (56.5%), ASCUS (34.4%), and HSIL (34.1%). HPV-6 was the most common genotype in normal cases (9.1%) and LSIL patients (18%). The prevalence of HPV positivity was significantly higher in cases with high-grade lesions (≥ HSIL) (64.2%) than in normal/LSIL (37.3%) (P = 0.033). The rate of HR-HPV infection was significantly higher in ≥ HSIL cases (61.2%) than normal/LSIL (27.9%) (P = 0.003). Conclusions This study describes robust information on the distribution of HPV genotypes among Iranian women with and without cervical lesions. The present data

  20. Role of hepatitis B virus genotype D & its mutants in occult hepatitis B infection

    PubMed Central

    Sengupta, Sonali; Panda, Subrat Kumar; Acharya, Subrat Kumar; Durgapal, Hemlata

    2013-01-01

    Background & objectives: Non-detection of hepatitis B virus (HBV) envelope protein (hepatitis B surface antigen, HBsAg) in a chronically HBV infected individual has been described as occult infection. One possible reason for this phenotype is alteration in large (L-HBsAg) to small (S-HBsAg) envelope protein ratio associated with reduced or non secretion of HBsAg. This results in quantitative levels of serum HBsAg below the detection limit of enzyme immunoassays. Genotype D of HBV has a characteristic 33 nucleotide (nt) deletion upstream of the pre-S2/S promoter. This deletion may reduce HBsAg secretion in occult infection patients infected with genotype D HBV. Additional deletions in the pre-S2/S promoter may further aggravate reduced HBsAg secretion in patients infected with genotype D HBV. Thus, the aim of the present study was to determine the role of genotype D specific 33nt deletion and additional pre-S2/S promoter deletions in causing reduced or no secretion of HBsAg, in occult infection. Since these deletions overlap virus polymerase, their effect on virus replication was also investigated. Methods: We examined the in vitro expression of HBsAg, ratio of cure and ‘e’ antigen (HBcAg/HBeAg), their secretion and virus replication, using overlength 1.3 mer/1.86 mer genotype A replicons, and genotype D replicons with and without additional pre-S2/S promoter deletions from cases of occult infection. Results: Genotype D replicon showed a decrease in HBsAg secretion compared to the wild-type genotype A. Genotype D replicons carrying additional pre-S2/S promoter deletions, showed further reduction in HBsAg secretion, demonstrated presence of intracellular HBcAg/HBeAg, virus replication intermediates and ‘e’ antigen secretion. Interpretation & conclusions: The characteristic 33 nt deletion of genotype D HBV reduces HBsAg secretion. Additional pre-S2/S promoter deletions may further diminish HBsAg secretion, leading to occult infection. Pre-S2/S promoter

  1. Influence of CYP2C9 Genotype on warfarin dose among African American and European Americans.

    PubMed

    Limdi, Na; Goldstein, Ja; Blaisdell, Ja; Beasley, Tm; Rivers, Ca; Acton, Rt

    2007-05-01

    BACKGROUND: Cytochrome P4502C9 (CYP2C9) plays a vital role in drug metabolism. There has been an increased effort to identify polymorphisms within the gene and determine their clinical consequences. However, most of these efforts have f