41 results on '"Zeichhardt, H."'
Search Results
2. Coxsackievirus B3 and adenovirus infections of cardiac cells are efficiently inhibited by vector-mediated RNA interference targeting their common receptor
- Author
-
Fechner, H, Pinkert, S, Wang, X, Sipo, I, Suckau, L, Kurreck, J, Dörner, A, Sollerbrant, K, Zeichhardt, H, Grunert, H-P, Vetter, R, Schultheiss, H-P, and Poller, W
- Published
- 2007
- Full Text
- View/download PDF
3. Treatment of coxsackievirus-B3-infected BALB/c mice with the soluble coxsackie adenovirus receptor CAR4/7 aggravates cardiac injury
- Author
-
Dörner, A., Grunert, H.-P., Lindig, V., Chandrasekharan, K., Fechner, H., Knowlton, K. U., Isik, A., Pauschinger, M., Zeichhardt, H., and Schultheiss, H.-P.
- Published
- 2006
- Full Text
- View/download PDF
4. The expression of Protease-Activated Receptor (PAR) 2 reduces the IFNbeta expression in the heart
- Author
-
Weithaeuser, A., primary, Bobbert, P., additional, Rauch, B., additional, Zeichhardt, H., additional, Schultheiss, H. P., additional, and Rauch, U., additional
- Published
- 2013
- Full Text
- View/download PDF
5. Inhibition of picornaviruses by means of RNA interference
- Author
-
Rothe, D., primary, Werk, D., additional, Dutkiewicz, M., additional, Schubert, S., additional, Grunert, H.-P., additional, Zeichhardt, H., additional, Erdmann, V. A., additional, Fechner, H., additional, and Kurreck, J., additional
- Published
- 2008
- Full Text
- View/download PDF
6. Inhibition of coxsackievirus B3 by RNA interference (RNAi)
- Author
-
Kurreck, J, primary, Schubert, S, additional, Werk, D, additional, Rothe, D, additional, Grunert, HP, additional, Sipo, I, additional, Fechner, H, additional, Poller, W, additional, Erdmann, VA, additional, and Zeichhardt, H, additional
- Published
- 2006
- Full Text
- View/download PDF
7. Prevention of cardiac dysfunction in acute coxsackievirus B3 cardiomyopathy by inducible expression of a soluble coxsackievirus-adenovirus receptor.
- Author
-
Pinkert S, Westermann D, Wang X, Klingel K, Dörner A, Savvatis K, Grössl T, Krohn S, Tschöpe C, Zeichhardt H, Kotsch K, Weitmann K, Hoffmann W, Schultheiss HP, Spiller OB, Poller W, and Fechner H
- Published
- 2009
- Full Text
- View/download PDF
8. Architecture of the Escherichia coli ribosome as determined by immune electron microscopy.
- Author
-
Tischendorf, G W, Zeichhardt, H, and Stöffler, G
- Abstract
Binding sites for antibodies specific to nineteen of the twenty-one ribosomal proteins from the 30S subunit of E. coli ribosomes have been localized on the surface of the 30S ribosomal subunit by immune electron microscopy. The locations of 13 ribosomal proteins from the 50S subunit were similarily assessed. The arrangement of these proteins is illustrated in three-dimensional models of the 30S and 50S ribosomal subunits and of 70S ribosomes. With specific antibodies to six proteins of the 30S subunit we found only one attachment point for each protein. Antibodies against each of nine of the proteins attached at two separate sites that were separated by various distances. Four further proteins were exposed at three or four sites for antibody binding. Altogether eight to ten of the 19 proteins of the 30S subunit have shown antibody attachment sites at remote points on the surface of the ribosome, at distances which are incompatible with globular shapes; these proteins must therefore have elongated or fibrous structures within the ribosome. On the other hand, only two proteins of the 50S subunit, namely L11 and L18, have so far revealed two separated antibody binding sites; proteins L7/L12 occurred, however, at multiple sites.
- Published
- 1975
- Full Text
- View/download PDF
9. Dense Particles and Slow Sedimenting Particles Produced by Ultraviolet Irradiation of Poliovirus
- Author
-
Wetz, K., primary, Zeichhardt, H., additional, Willingmann, P., additional, and Habermehl, K.-O., additional
- Published
- 1983
- Full Text
- View/download PDF
10. Mouse Elberfeld (ME) Virus Determines the Cell Surface Alterations when Mixedly Infecting Poliovirus-infected Cells
- Author
-
Zeichhardt, H., primary, Schlehofer, J. R., additional, Wetz, K., additional, Hampl, H., additional, and Habermehl, K.-O., additional
- Published
- 1982
- Full Text
- View/download PDF
11. A Poliovirus-induced Cytoplasmic Membrane Complex is Exploited by the RNA Polymerase of Superinfecting Mouse Elberfeld (ME) Virus
- Author
-
Zeichhardt, H., primary, Habermehl, K.-O., additional, and Wetz, K., additional
- Published
- 1983
- Full Text
- View/download PDF
12. Entry of Poliovirus Type 1 and Mouse Elberfeld (ME) Virus into HEp-2 Cells: Receptor-mediated Endocytosis and Endosomal or Lysosomal Uncoating
- Author
-
Zeichhardt, H., primary, Wetz, K., additional, Willingmann, P., additional, and Habermehl, K.-O., additional
- Published
- 1985
- Full Text
- View/download PDF
13. Modification and Exploitation of a Poliovirus-induced Membrane Complex by Superinfecting ME Virus
- Author
-
Zeichhardt, H., primary, Habermehl, K.-O., additional, and Diefenthal, W., additional
- Published
- 1981
- Full Text
- View/download PDF
14. Induktion neutralisierender Antikörper gegen transmembrane Hüllproteine von Retroviren
- Author
-
Fiebig, Uwe, Krüger, D. H., Kurth, R., and Zeichhardt, H.
- Subjects
32 Biologie ,610 Medizin ,HIV ,Impfstoffentwicklung ,Induktion ,neutralisierende Antikörper ,vaccine development ,ddc:570 ,PERV ,Hüllproteine ,Antikörper ,570 Biowissenschaften, Biologie ,neutralizing antibodies ,Retroviren ,ddc:610 ,WF 4780 ,XD 8000 - Abstract
Die Transplantation von porzinen Organen könnte eine Lösung des akuten Mangels von Allotransplantaten in der Transplantationsmedizin darstellen. Bevor die Xenotransplantation klinische Realität werden kann, sind jedoch zahlreiche Hürden zu überwinden. Insbesondere die mögliche Übertragung porziner endogener Retroviren (PERVs), die integraler Bestandteil des porzinen Genoms sind, stellt ein mikrobiologisches Risiko dar. PERVs können humane Zellen in vitro produktiv infizieren. Mögliche Strategien zur Abwehr von Xenosen sind die Verwendung von PERV-knockout Tieren oder die Entwicklung eines effektiven Impfstoffes, durch den der Transplantatrezipient vor einer möglichen Übertragung geschützt werden kann. Dazu wurden Antiseren gegen die Hauptstrukturproteine von PERV generiert. Es konnte gezeigt werden, dass in Ziegen und Ratten durch Immunisierung mit der rekombinant generiereten Ektodomäne des transmembranen Hüllprotein p15E neutralisierende Antikörper induziert werden konnten. Die Epitopkartierung der induzierten Antikörper zeigt eine Bindung an eine Domäne im N-terminalen, nahe des Fusionspeptids (E1, GPQQLEK) und eine Domäne im C-terminalen, membranproximalen (E2, FEGWFN) Bereich der p15E-Ektodomäne. Diese Sequenzen sind in allen PERVs identisch und innerhalb der Gammaretroviren hochkonserviert. Aus AIDS-Patienten isolierte neutralisierende Antikörper (mAb2F5: ELDKWA, mAb4E10: LWNWFN) binden ebenfalls an den C-terminalen Bereich der Ektodomäne des Transmembranproteins gp41. Der Bindungsmechannismus dieser Antikörper wurde in ELISA-Experimenten und in vitro-Inhibitionsassays analysiert. Die Ergebnisse legen die Bindung eines Konformationsepitopes nahe, das aus der E1 und der E2 Domäne gebildet wird. Die Aufklärung des Bindungsmechnismus breit neutralisierender Antikörper gegen Transmembranproteine von Retroviren könnte die Basis für neue Impfstoffansätze darstellen., Porcine xenotransplants may offer a potential solution to the problem posed by the limited supply of allotransplants. However, xenotransplantation may be associated with the risk of transmission of microorganisms, in particular of porcine endogenous retroviruses (PERVs) that are an integral part of the porcine genome and able to infect human cells in vitro. Possible strategies to prevent virus transmission include the development of PERV knockout animals or of effective vaccines. When antisera prepared against the main structural proteins of PERV were screened, goat and rat antisera against the recombinant ectodomain of the transmembrane envelope protein p15E were found to neutralize PERV infectivity. Epitope mapping using overlapping peptides revealed two epitopes, one near the fusion peptide (E1, GPQQLEK) and the other near the transmembrane domain (E2, FEGWFN). These sequences are identical for all PERVs and are highly conserved among all gammaretroviruses. Interestingly, neutralizing antibodies isolated from AIDS patients that recognize regions partially homologous with E2 (mAb4E10, LWNWFN) or located in close proximity to E2 (mAb2F5, ELDKWA) are known to neutralize a broad range of HIV-1 strains. The binding mechanisms of these HIV neutralizing antibodies were analyzed in ELISA experiments and in vitro inhibition assays. The results indicate that the two most broadly reactive HIV-1 envelope gp41 human mAbs are specific for a discontinuous epitope composed of the E1 and the E2 domain. If so, these two transmembrane protein domains in different retroviruses act as effective targets for neutralizing antibodies and may provide the basis for effective antiretroviral vaccines.
- Published
- 2008
15. Do not blindly trust negative diagnostic test results!
- Author
-
Buchta C, Zeichhardt H, Osterman A, Perrone LA, and Griesmacher A
- Subjects
- Humans, Diagnostic Tests, Routine, Trust
- Abstract
Competing Interests: HZ declares that he was the majority owner and managing director of GBD Gesellschaft für Biotechnologische Diagnostik mbH, Berlin and is the owner and managing director of IQVD GmbH, Institut für Qualitätssicherung, Berlin. LAP received support from the Donald B Rix Family Foundation. All other authors declare no competing interests.
- Published
- 2024
- Full Text
- View/download PDF
16. Hantavirus Disease Cluster Caused by Seoul Virus, Germany.
- Author
-
Hofmann J, Ulrich RG, Mehl C, Drewes S, Esser J, Loyen M, Zeichhardt H, Schoppmeyer K, Essen L, Güthoff W, and Krüger DH
- Subjects
- Animals, Rats, Disease Hotspot, Germany epidemiology, Europe, Seoul virus genetics, Orthohantavirus, RNA Viruses
- Abstract
A cluster of 3 persons in Germany experienced hantavirus disease with renal insufficiency. Reverse transcription PCR-based genotyping revealed infection by Seoul hantavirus transmitted from pet rats. Seoul virus could be responsible for disease clusters in Europe, and infected pet rats should be considered a health threat.
- Published
- 2024
- Full Text
- View/download PDF
17. Correlation of SARS-CoV-2 RNA and nucleocapsid concentrations in samples used in INSTAND external quality assessment schemes.
- Author
-
Valiente E, Falak S, Kummrow A, Kammel M, Corman VM, Macdonald R, and Zeichhardt H
- Subjects
- Humans, SARS-CoV-2 genetics, Nucleocapsid, RNA, Viral genetics, COVID-19 diagnosis
- Abstract
Objective: In routine clinical laboratories, severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection is determined by reverse-transcription PCR (RT-PCR). In the COVID pandemic, a wide range of antigen detection tests were also in high demand. We investigated the correlation between SARS-CoV-2 NCap antigen and N gene concentration by analyzing samples from several INSTAND external quality assessment (EQA) schemes starting in March 2021. The absolute N gene concentration was measured using reverse transcriptase digital PCR (RT-dPCR) as reference value. Moreover, the performance of five commercial ELISA tests using an EQA inactivated SARS-CoV-2 sample at different concentrations was assessed on the basis of these reference values., Results: Quantitative ELISA and RT-dPCR results showed a good correlation between SARS-CoV-2 NCap antigen and RNA concentration, but this correlation varies among SARS-CoV-2 isolates. A direct correlation between SARS-CoV-2 NCap antigen concentration and genome concentration should not be generally assumed., Conclusion: Further correlation studies between SARS-CoV-2 RNA and NCap antigen concentrations are needed, particularly in clinical samples and for emerging SARS-CoV-2 variants, to support the monitoring and improvement of antigen testing., (© 2023. BioMed Central Ltd., part of Springer Nature.)
- Published
- 2023
- Full Text
- View/download PDF
18. Results of German external quality assessment schemes for SARS-CoV-2 antigen detection.
- Author
-
Vierbaum L, Wojtalewicz N, Grunert HP, Zimmermann A, Scholz A, Goseberg S, Kaiser P, Duehring U, Drosten C, Corman V, Niemeyer D, Rabenau HF, Obermeier M, Nitsche A, Michel J, Puyskens A, Huggett JF, O'Sullivan DM, Busby E, Cowen S, Vallone PM, Cleveland MH, Falak S, Kummrow A, Schellenberg I, Zeichhardt H, and Kammel M
- Subjects
- Chlorocebus aethiops, Animals, Humans, Pandemics, Vero Cells, Immunologic Tests, Sensitivity and Specificity, SARS-CoV-2, COVID-19 diagnosis, COVID-19 epidemiology
- Abstract
The COVID-19 pandemic illustrated the important role of diagnostic tests, including lateral flow tests (LFTs), in identifying patients and their contacts to slow the spread of infections. INSTAND performed external quality assessments (EQA) for SARS-CoV-2 antigen detection with lyophilized and chemically inactivated cell culture supernatant of SARS-CoV-2 infected Vero cells. A pre-study demonstrated the suitability of the material. Participants reported qualitative and/or quantitative antigen results using either LFTs or automated immunoassays for five EQA samples per survey. 711 data sets were reported for LFT detection in three surveys in 2021. This evaluation focused on the analytical sensitivity of different LFTs and automated immunoassays. The inter-laboratory results showed at least 94% correct results for non-variant of concern (VOC) SARS-CoV-2 antigen detection for viral loads of ≥ 4.75 × 10
6 copies/mL and SARS-CoV-2 negative samples. Up to 85% had success for a non-VOC viral load of ~ 1.60 × 106 copies/mL. A viral load of ~ 1.42 × 107 copies/mL of the Delta VOC was reported positive in > 96% of results. A high specificity was found with almost 100% negative SARS-CoV-2 antigen results for HCoV 229E and HCoV NL63 positive samples. Quantitative results correlated with increasing SARS-CoV-2 viral load but showed a broad scatter. This study shows promising SARS-CoV-2 antigen test performance of the participating laboratories, but further investigations with the now predominant Omicron VOC are needed., (© 2023. Springer Nature Limited.)- Published
- 2023
- Full Text
- View/download PDF
19. Design of external quality assessment schemes and definition of the roles of their providers in future epidemics.
- Author
-
Buchta C, Zeichhardt H, Aberle SW, Camp JV, Görzer I, Weseslindtner L, Puchhammer-Stöckl E, Huf W, Benka B, Allerberger F, Mielke M, Griesmacher A, Müller MM, Schellenberg I, and Kammel M
- Subjects
- Humans, Reproducibility of Results, SARS-CoV-2 genetics, Laboratories, Pandemics, COVID-19 diagnosis, COVID-19 epidemiology
- Abstract
During an epidemic, individual test results form the basis of epidemiological indicators such as case numbers or incidence. Therefore, the accuracy of measures derived from these indicators depends on the reliability of individual results. In the COVID-19 pandemic, monitoring and evaluating the performance of the unprecedented number of testing facilities in operation, and novel testing systems in use, was urgently needed. External quality assessment (EQA) schemes are unique sources of data reporting on testing performance, and their providers are recognised contacts and support for test facilities (for technical-analytical topics) and health authorities (for planning the monitoring of infection diagnostics). To identify information provided by SARS-CoV-2 genome detection EQA schemes that is relevant for public health microbiology, we reviewed the current literature published in PubMed between January, 2020, and July, 2022. We derived recommendations for EQA providers and their schemes for best practices to monitor pathogen-detection performance in future epidemics. We also showed laboratories, test facilities, and health authorities the information and benefits they can derive from EQA data, and from the non-EQA services of their providers., Competing Interests: Declaration of interests CB is chairman of the executive board of the European Organisation for External Quality Assurance Providers in Laboratory Medicine 2020–23. HZ was majority owner of Gesellschaft für Biotechnologische Diagnostik (until November, 2022) and is owner and managing director of Institut für Qualitätssicherung in der Virusdiagnostik. AG is president of the Austrian Association for Quality Assurance and Standardization of Medical and Diagnostic Tests. All other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2023
- Full Text
- View/download PDF
20. Ignoring SARS-CoV-2 testing performance during COVID-19.
- Author
-
Buchta C, Zeichhardt H, Griesmacher A, Schellenberg I, and Kammel M
- Subjects
- Humans, SARS-CoV-2, COVID-19 Testing, Sensitivity and Specificity, COVID-19
- Abstract
Competing Interests: HZ is majority owner of Gesellschaft für Biotechnologische Diagnostik; and owner and managing director of Institut für Qualitätssicherung in der Virusdiagnostik. All other authors declare no competing interests.
- Published
- 2023
- Full Text
- View/download PDF
21. Results of the first German external quality assessment scheme for the detection of monkeypox virus DNA.
- Author
-
Vierbaum L, Wojtalewicz N, Kaufmann A, Goseberg S, Kaiser P, Grunert HP, Dühring U, Zimmermann A, Scholz A, Michel J, Nitsche A, Rabenau HF, Obermeier M, Schellenberg I, Zeichhardt H, and Kammel M
- Subjects
- Humans, Monkeypox virus genetics, SARS-CoV-2 genetics, COVID-19, Mpox (monkeypox), Orthopoxvirus
- Abstract
Background: In May 2022, the monkeypox virus (MPXV) spread into non-endemic countries and the global community was quick to test the lessons learned from the SARS-CoV-2 pandemic. Due to its symptomatic resemblance to other diseases, like the non-pox virus varicella zoster (chickenpox), polymerase chain reaction methods play an important role in correctly diagnosing the rash-causing pathogen. INSTAND quickly established a new external quality assessment (EQA) scheme for MPXV and orthopoxvirus (OPXV) DNA detection to assess the current performance quality of the laboratory tests., Methods: We analyzed quantitative and qualitative data of the first German EQA for MPXV and OPXV DNA detection. The survey included one negative and three MPXV-positive samples with different MPX viral loads. The threshold cycle (Ct) or other measures defining the quantification cycle (Cq) were analyzed in an assay-specific manner. A Passing Bablok fit was used to investigate the performance at laboratory level., Results: 141 qualitative datasets were reported by 131 laboratories for MPXV detection and 68 qualitative datasets by 65 laboratories for OPXV detection. More than 96% of the results were correctly identified as negative and more than 97% correctly identified as positive. An analysis of the reported Ct/Cq values showed a large spread of these values of up to 12 Ct/Cq. Nevertheless, there is a good correlation of results for the different MPXV concentrations at laboratory level. Only a few quantitative results in copies/mL were reported (MPXV: N = 5; OPXV: N = 2), but the results correlated well with the concentration differences between the EQA samples, which were to a power of ten each., Conclusion: The EQA results show that laboratories performed well in detecting both MPXV and OPXV. However, Ct/Cq values should be interpreted with caution when conclusions are drawn about the viral load as long as metrological traceability is not granted., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: HZ declares that he was majority owner of GBD Gesellschaft fuer Biotechnologische Diagnostik mbH, Berlin, and owner and managing director of IQVD GmbH - Institut fuer Qualitaetssicherung, Berlin. HPG declares that he was minority owner of GBD Gesellschaft fuer Biotechnologische Diagnostik mbH, Berlin. HZ and HPG declare that they were managing directors of GBD Gesellschaft fuer Biotechnologische Diagnostik mbH, Berlin, during the study. This does not alter our adherence to PLoS One policies on sharing data and materials. All other authors have declared that no competing interests exist., (Copyright: © 2023 Vierbaum et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
- Published
- 2023
- Full Text
- View/download PDF
22. The Coronavirus Standards Working Group's roadmap for improved population testing.
- Author
-
Mercer T, Almond N, Crone MA, Chain PSG, Deshpande A, Eveleigh D, Freemont P, Fuchs S, Garlick R, Huggett J, Kammel M, Li PE, Milavec M, Marlowe EM, O'Sullivan DM, Page M, Pestano GA, Suliman S, Simen B, Sninsky JJ, Sopchak L, Tato CM, Vallone PM, Vandesompele J, White TJ, Zeichhardt H, and Salit M
- Subjects
- Coronavirus
- Published
- 2022
- Full Text
- View/download PDF
23. Harmonization of Multiple SARS-CoV-2 Reference Materials Using the WHO IS (NIBSC 20/136): Results and Implications.
- Author
-
Windsor WJ, Roell Y, Tucker H, Cheng CA, Suliman S, Peek LJ, Pestano GA, Lee WT, Zeichhardt H, Lamb MM, Kammel M, Wang H, Kedl R, Rester C, Morrison TE, Davenport BJ, Carson K, Yates J, Howard K, Kulas K, Walt DR, Dafni A, Taylor D, and Chu M
- Abstract
Background: There is an urgent need for harmonization between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology platforms and assays prior to defining appropriate correlates of protection and as well inform the development of new rapid diagnostic tests that can be used for serosurveillance as new variants of concern (VOC) emerge. We compared multiple SARS-CoV-2 serology reference materials to the WHO International Standard (WHO IS) to determine their utility as secondary standards, using an international network of laboratories with high-throughput quantitative serology assays. This enabled the comparison of quantitative results between multiple serology platforms., Methods: Between April and December 2020, 13 well-characterized and validated SARS-CoV-2 serology reference materials were recruited from six different providers to qualify as secondary standards to the WHO IS. All the samples were tested in parallel with the National Institute for Biological Standards and Control (NIBSC) 20/136 and parallel-line assays were used to calculate the relevant potency and binding antibody units., Results: All the samples saw varying levels of concordance between diagnostic methods at specific antigen-antibody combinations. Seven of the 12 candidate materials had high concordance for the spike-immunoglobulin G (IgG) analyte [percent coefficient of variation (%CV) between 5 and 44%]., Conclusion: Despite some concordance between laboratories, qualification of secondary materials to the WHO IS using arbitrary international units or binding antibody units per milliliter (BAU/ml) does not provide any benefit to the reference materials overall, due to the lack of consistent agreeable international unit (IU) or BAU/ml conversions between laboratories. Secondary standards should be qualified to well-characterized reference materials, such as the WHO IS, using serology assays that are similar to the ones used for the original characterization of the WHO IS., Competing Interests: GP and LP was employed by Biodesix, Inc. HZ and MK was employed by INSTAND e.V., IQVD GmbH, and GBD Gesellschaft fuer Biotechnologische Diagnostik mbH. HW was employed by Thermo Fisher Scientific. AD and DT were employed by company Oneworld Accuracy. The authors declare that this study received funding from the Bill & Melinda Gates Foundation. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Windsor, Roell, Tucker, Cheng, Suliman, Peek, Pestano, Lee, Zeichhardt, Lamb, Kammel, Wang, Kedl, Rester, Morrison, Davenport, Carson, Yates, Howard, Kulas, Walt, Dafni, Taylor and Chu.)
- Published
- 2022
- Full Text
- View/download PDF
24. Evaluation of the diagnostic capacities for emerging arboviral diseases in the international network MediLabSecure from 2014 to 2018 - Importance of external quality assessments.
- Author
-
Mikaty G, Matheus S, Donoso Mantke O, McCulloch E, Zeichhardt H, and Manuguerra JC
- Subjects
- Humans, Arbovirus Infections diagnosis, Arbovirus Infections epidemiology, Arboviruses, Chikungunya Fever, Zika Virus, Zika Virus Infection
- Abstract
Background: Emerging infectious diseases pose an increasing threat to all nations around the world, including to developed countries. By definition, because they are rare or unknown, public health systems are not well prepared against these emerging diseases. To be fully prepared, countries must have implemented surveillance systems to monitor rare or unusual sanitary events., Methods: The capacity of diagnostic laboratories is a key component of surveillance systems since they are in charge of identifying the pathogens responsible for outbreaks in a timely manner. The MediLabSecure project aims at implementing a comprehensive surveillance system for vector-borne diseases around the Mediterranean and Black Sea regions. From 2014 to 2018, the human-virology group of MediLabSecure notably supported the implementation of molecular diagnostic capacities for eight arboviruses and one coronavirus in 19 laboratories of its network through sharing of protocols and reagents, and technical training of the scientific staff of beneficiary laboratories., Results: We report the results of External Quality Assessments for four of these viruses to assess the efficiency of the diagnostic for these threats emerging in the geographic area. The results for these EQA demonstrate the success of the project in the implementation of diagnostic technics for the identification of Dengue, Chikungunya, Zika, and West Niles viruses in laboratories that did not have the capacity before. However, results also show that some work is still to be done to strengthen the newly acquired capacity., Conclusion: The MediLabSecure project deployed an effort to build an efficient capacity in identifying and survey the emergence of arboviruses in the Mediterranean area. Diagnostic technics were successfully implemented in many of the laboratories of the network, but the effort must be maintained over time to strengthen these capacities., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2022
- Full Text
- View/download PDF
25. RNA reference materials with defined viral RNA loads of SARS-CoV-2-A useful tool towards a better PCR assay harmonization.
- Author
-
Vierbaum L, Wojtalewicz N, Grunert HP, Lindig V, Duehring U, Drosten C, Corman V, Niemeyer D, Ciesek S, Rabenau HF, Berger A, Obermeier M, Nitsche A, Michel J, Mielke M, Huggett J, O'Sullivan D, Busby E, Cowen S, Vallone PM, Cleveland MH, Falak S, Kummrow A, Keller T, Schellenberg I, Zeichhardt H, and Kammel M
- Subjects
- COVID-19 epidemiology, COVID-19 virology, Genes, Viral, Germany epidemiology, Humans, Reproducibility of Results, COVID-19 diagnosis, COVID-19 Nucleic Acid Testing methods, Diagnostic Tests, Routine methods, RNA, Viral genetics, Reverse Transcriptase Polymerase Chain Reaction methods, SARS-CoV-2 genetics, Viral Load methods
- Abstract
SARS-CoV-2, the cause of COVID-19, requires reliable diagnostic methods to track the circulation of this virus. Following the development of RT-qPCR methods to meet this diagnostic need in January 2020, it became clear from interlaboratory studies that the reported Ct values obtained for the different laboratories showed high variability. Despite this the Ct values were explored as a quantitative cut off to aid clinical decisions based on viral load. Consequently, there was a need to introduce standards to support estimation of SARS-CoV-2 viral load in diagnostic specimens. In a collaborative study, INSTAND established two reference materials (RMs) containing heat-inactivated SARS-CoV-2 with SARS-CoV-2 RNA loads of ~107 copies/mL (RM 1) and ~106 copies/mL (RM 2), respectively. Quantification was performed by RT-qPCR using synthetic SARS-CoV-2 RNA standards and digital PCR. Between November 2020 and February 2021, German laboratories were invited to use the two RMs to anchor their Ct values measured in routine diagnostic specimens, with the Ct values of the two RMs. A total of 305 laboratories in Germany were supplied with RM 1 and RM 2. The laboratories were requested to report their measured Ct values together with details on the PCR method they used to INSTAND. This resultant 1,109 data sets were differentiated by test system and targeted gene region. Our findings demonstrate that an indispensable prerequisite for linking Ct values to SARS-CoV-2 viral loads is that they are treated as being unique to an individual laboratory. For this reason, clinical guidance based on viral loads should not cite Ct values. The RMs described were a suitable tool to determine the specific laboratory Ct for a given viral load. Furthermore, as Ct values can also vary between runs when using the same instrument, such RMs could be used as run controls to ensure reproducibility of the quantitative measurements., Competing Interests: The work from SF and AK was partially funded by EURAMET in the project 18HLT03 SEPTIMET. The funder had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. AK is sitting in ISO/TC212/JWG 6 that is currently developing a standard for quality practice for detection of SARS-CoV-2. Work for this committee had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. TK is the owner of ACOMED statistik. He declares that he has received a payment from INSTAND e.V. for statistical consulting. He has received further payments from INSTAND e.V. in other projects. HZ declares that he is majority owner and managing director of GBD Gesellschaft fuer Biotechnologische Diagnostik mbH, Berlin, and owner and managing director of IQVD GmbH - Institut fuer Qualitaetssicherung, Berlin. HPG declares that he is minority owner and managing director of GBD Gesellschaft für Biotechnologische Diagnostik mbH, Berlin. This does not alter our adherence to PLOS ONE policies on sharing data and materials. All other authors have declared that no competing interests exist.
- Published
- 2022
- Full Text
- View/download PDF
26. The Dangers of Using Cq to Quantify Nucleic Acid in Biological Samples: A Lesson From COVID-19.
- Author
-
Evans D, Cowen S, Kammel M, O'Sullivan DM, Stewart G, Grunert HP, Moran-Gilad J, Verwilt J, In J, Vandesompele J, Harris K, Hong KH, Storey N, Hingley-Wilson S, Dühring U, Bae YK, Foy CA, Braybrook J, Zeichhardt H, and Huggett JF
- Subjects
- Belgium, Humans, RNA, Viral analysis, Reproducibility of Results, Republic of Korea, SARS-CoV-2, Sensitivity and Specificity, United Kingdom, COVID-19 diagnosis, COVID-19 Nucleic Acid Testing standards, Nucleic Acids analysis
- Abstract
Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA quantities, measured by reverse transcription quantitative PCR (RT-qPCR), have been proposed to stratify clinical risk or determine analytical performance targets. We investigated reproducibility and how setting diagnostic cutoffs altered the clinical sensitivity of coronavirus disease 2019 (COVID-19) testing., Methods: Quantitative SARS-CoV-2 RNA distributions [quantification cycle (Cq) and copies/mL] from more than 6000 patients from 3 clinical laboratories in United Kingdom, Belgium, and the Republic of Korea were analyzed. Impact of Cq cutoffs on clinical sensitivity was assessed. The June/July 2020 INSTAND external quality assessment scheme SARS-CoV-2 materials were used to estimate laboratory reported copies/mL and to estimate the variation in copies/mL for a given Cq., Results: When the WHO-suggested Cq cutoff of 25 was applied, the clinical sensitivity dropped to about 16%. Clinical sensitivity also dropped to about 27% when a simulated limit of detection of 106 copies/mL was applied. The interlaboratory variation for a given Cq value was >1000 fold in copies/mL (99% CI)., Conclusion: While RT-qPCR has been instrumental in the response to COVID-19, we recommend Cq (cycle threshold or crossing point) values not be used to set clinical cutoffs or diagnostic performance targets due to poor interlaboratory reproducibility; calibrated copy-based units (used elsewhere in virology) offer more reproducible alternatives. We also report a phenomenon where diagnostic performance may change relative to the effective reproduction number. Our findings indicate that the disparities between patient populations across time are an important consideration when evaluating or deploying diagnostic tests. This is especially relevant to the emergency situation of an evolving pandemic., (© American Association for Clinical Chemistry 2021.)
- Published
- 2021
- Full Text
- View/download PDF
27. Cautionary Note on Contamination of Reagents Used for Molecular Detection of SARS-CoV-2.
- Author
-
Huggett JF, Benes V, Bustin SA, Garson JA, Harris K, Kammel M, Kubista M, McHugh TD, Moran-Gilad J, Nolan T, Pfaffl MW, Salit M, Shipley G, Vallone PM, Vandesompele J, Wittwer C, and Zeichhardt H
- Subjects
- COVID-19 virology, Diagnostic Errors, Humans, Limit of Detection, Nasopharynx virology, RNA, Viral genetics, RNA, Viral metabolism, Reverse Transcriptase Polymerase Chain Reaction, SARS-CoV-2 isolation & purification, Specimen Handling standards, COVID-19 diagnosis, Indicators and Reagents chemistry, SARS-CoV-2 genetics
- Published
- 2020
- Full Text
- View/download PDF
28. Targeting Highly Structured RNA by Cooperative Action of siRNAs and Helper Antisense Oligomers in Living Cells.
- Author
-
Dutkiewicz M, Ojdowska A, Kuczynski J, Lindig V, Zeichhardt H, Kurreck J, and Ciesiołka J
- Subjects
- 5' Untranslated Regions genetics, Base Sequence, Enterovirus B, Human drug effects, Enterovirus Infections genetics, Enterovirus Infections virology, Gene Silencing, HeLa Cells, Humans, Molecular Sequence Data, Nucleic Acid Conformation, Ribonuclease H genetics, Antiviral Agents pharmacology, Enterovirus B, Human genetics, Oligonucleotides, Antisense pharmacology, RNA, Small Interfering pharmacology, RNA, Viral genetics
- Abstract
RNA target accessibility is one of the most important factors limiting the efficiency of RNA interference-mediated RNA degradation. However, targeting RNA viruses in their poorly accessible, highly structured regions can be advantageous because these regions are often conserved in sequence and thus less prone to viral escape. We developed an experimental strategy to attack highly structured RNA by means of pairs of specifically designed small interfering RNAs and helper antisense oligonucleotides using the 5' untranslated region (5'UTR) of coxsackievirus B3 as a model target. In the first step, sites accessible to hybridization of complementary oligonucleotides were identified using two mapping methods with random libraries of short DNA oligomers. Subsequently, the accessibility of the mapped regions for hybridization of longer DNA 16-mers was confirmed by an RNase H assay. Using criteria for the design of efficient small interfering RNAs (siRNA) and a secondary structure model of the viral 5'UTR, several DNA 19-mers were designed against partly double-stranded RNA regions. Target sites for DNA 19-mers were located opposite the sites which had been confirmed as accessible for hybridization. Three pairs of DNA 19-mers and the helper 2'-O-methyl-16-mers were able to effectively induce RNase H cleavage in vitro. For cellular assays, the DNA 19-mers were replaced by siRNAs, and the corresponding three pairs of siRNA-helper oligomer tools were found to target 5'UTR efficiently in a reporter construct in HeLa cells. Addition of the helper oligomer improved silencing capacity of the respective siRNA. We assume that the described procedure will generally be useful for designing of nucleic acid-based tools to silence highly structured RNA targets.
- Published
- 2015
- Full Text
- View/download PDF
29. Standardization of Nucleic Acid Tests for Clinical Measurements of Bacteria and Viruses.
- Author
-
Pavšič J, Devonshire AS, Parkes H, Schimmel H, Foy CA, Karczmarczyk M, Gutiérrez-Aguirre I, Honeyborne I, Huggett JF, McHugh TD, Milavec M, Zeichhardt H, and Žel J
- Subjects
- Bacterial Infections microbiology, Bacterial Load methods, Humans, Molecular Diagnostic Techniques methods, Reference Standards, Viral Load methods, Virus Diseases virology, Bacteria isolation & purification, Bacterial Load standards, Molecular Diagnostic Techniques standards, Viral Load standards, Viruses isolation & purification
- Abstract
Nucleic acid-based tests for infectious diseases currently used in the clinical laboratory and in point-of-care devices are diverse. Measurement challenges associated with standardization of quantitative viral load testing are discussed in relation to human cytomegalovirus, BK virus, and Epstein-Barr virus, while the importance of defining the performance of qualitative methods is illustrated with Mycobacterium tuberculosis and influenza virus. The development of certified reference materials whose values are traceable to higher-order standards and reference measurement procedures, using, for instance, digital PCR, will further contribute to the understanding of analytical performance characteristics and promote clinical data comparability., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)
- Published
- 2015
- Full Text
- View/download PDF
30. Protease-activated receptor-2 regulates the innate immune response to viral infection in a coxsackievirus B3-induced myocarditis.
- Author
-
Weithauser A, Bobbert P, Antoniak S, Böhm A, Rauch BH, Klingel K, Savvatis K, Kroemer HK, Tschope C, Stroux A, Zeichhardt H, Poller W, Mackman N, Schultheiss HP, and Rauch U
- Subjects
- Animals, Antibodies, Viral analysis, Biopsy, Blotting, Western, Disease Models, Animal, Enterovirus Infections virology, Gene Expression Regulation, Immunohistochemistry, Male, Mice, Mice, Inbred C57BL, Myocarditis metabolism, Myocarditis virology, Myocardium pathology, RNA genetics, Real-Time Polymerase Chain Reaction, Receptor, PAR-2 genetics, Toll-Like Receptor 3 biosynthesis, Toll-Like Receptor 3 genetics, Enterovirus B, Human immunology, Enterovirus Infections immunology, Immunity, Innate, Myocarditis immunology, Myocardium enzymology, Receptor, PAR-2 biosynthesis
- Abstract
Objectives: This study sought to evaluate the role of protease-activated receptor-2 (PAR2) in coxsackievirus B3 (CVB3)-induced myocarditis., Background: An infection with CVB3 leads to myocarditis. PAR2 modulates the innate immune response. Toll-like receptor-3 (TLR3) is crucial for the innate immune response by inducing the expression of the antiviral cytokine interferon-beta (IFNβ)., Methods: To induce myocarditis, wild-type (wt) and PAR2 knockout (ko) mice were infected with 10(5) plaque-forming units CVB3. Mice underwent hemodynamic measurements with a 1.2-F microconductance catheter. Wt and PAR2ko hearts and cardiac cells were analyzed for viral replication and immune response with plaque assay, quantitative polymerase chain reaction, Western blot, and immunohistochemistry., Results: Compared with wt mice, PAR2ko mice and cardiomyocytes exhibited a reduced viral load and developed no myocarditis after infection with CVB3. Hearts and cardiac fibroblasts from PAR2ko mice expressed higher basal levels of IFNβ than wt mice did. Treatment with CVB3 and polyinosinic:polycytidylic acid led to higher IFNβ expression in PAR2ko than in wt fibroblasts and reduced virus replication in PAR2ko fibroblasts was abrogated by neutralizing IFNβ antibody. Overexpression of PAR2 reduced the basal IFNβ expression. Moreover, a direct interaction between PAR2 and Toll-like receptor 3 was observed. PAR2 expression in endomyocardial biopsies of patients with nonischemic cardiomyopathy was positively correlated with myocardial inflammation and negatively with IFNβ expression and left ventricular ejection fraction., Conclusions: PAR2 negatively regulates the innate immune response to CVB3 infection and contributes to myocardial dysfunction. The antagonism of PAR2 is of therapeutic interest to strengthen the antiviral response after an infection with a cardiotropic virus., (Copyright © 2013 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
31. Spiegelzymes: sequence specific hydrolysis of L-RNA with mirror image hammerhead ribozymes and DNAzymes.
- Author
-
Wyszko E, Szymański M, Zeichhardt H, Müller F, Barciszewski J, and Erdmann VA
- Subjects
- Animals, Base Sequence, COS Cells, Chlorocebus aethiops, DNA, Catalytic blood, DNA, Catalytic chemistry, Deoxyribonucleases metabolism, Humans, Hydrolysis, Models, Molecular, Nucleic Acid Conformation, RNA chemistry, RNA Stability, RNA, Catalytic blood, RNA, Catalytic chemistry, Ribonucleases metabolism, DNA, Catalytic metabolism, RNA metabolism, RNA, Catalytic metabolism
- Abstract
In this manuscript we describe for the first time mirror image catalytic nucleic acids (Spiegelzymes), which hydrolyze sequence specifically L-ribonucleic acid molecules. The mirror image nucleic acid ribozymes designed are based upon the known hammerhead ribozyme and DNAzyme structures that contain L-ribose or L-deoxyribose instead of the naturally occurring D-ribose or D-deoxyribose, respectively. Both Spiegelzymes show similar hydrolytic activities with the same L-RNA target molecules and they also exhibit extra ordinary stabilities when tested with three different human sera. In this respect they are very similar to Spiegelmers (mirror image aptamers), which we had previously developed and for which it has been shown that they are non-toxic and non-immunogenic. Since we are also able to demonstrate that the hammerhead and DNAzyme Spiegelzymes can also hydrolyze mirror image oligonucleotide sequences, like they occur in Spiegelmers, in vivo, it seems reasonable to assume that Spiegelzymes may in principle be used as an antidote against Spiegelmers. Since the Spiegelzymes contain the same building blocks as the Spiegelmers, it can be expected that they will have similar favorable biological characteristics concerning toxicity and immunogenety. In trying to understand the mechanism of action of the Spiegelzymes described in this study, we have initiated for the first time a model building system with L-nucleic acids. The models for L-hammerhead ribozyme and L-DNAzyme interaction with the same L-RNA target will be presented.
- Published
- 2013
- Full Text
- View/download PDF
32. The mitochondrial respiratory chain has a critical role in the antiviral process in Coxsackievirus B3-induced myocarditis.
- Author
-
Ebermann L, Wika S, Klumpe I, Hammer E, Klingel K, Lassner D, Völker U, Erben U, Zeichhardt H, Schultheiss HP, and Dörner A
- Subjects
- Animals, Apoptosis, Disease Resistance, Electron Transport Complex I physiology, Heart virology, Male, Mice, Mice, Inbred C57BL, Oxidative Stress, Viral Load, Coxsackievirus Infections immunology, Electron Transport physiology, Enterovirus B, Human, Mitochondria, Heart physiology, Myocarditis immunology
- Abstract
Well-established differences in Coxsackievirus B3 (CVB3) elimination in resistant C57BL/6 and permissive A.SW/SnJ mice provide suitable models for studying the significance of the link between mitochondrial respiratory chain (RC), antioxidative stress components and mitochondrion-related apoptosis in the context of myocardial virus elimination. Distinct myocardial CVB3 titer in C57BL/6 (2.5 ± 1.4 × 10(4) plaque-forming units (p.f.u.)/g tissue) and A.SW/SnJ mice (1.4 ± 0.8 × 10(7) p.f.u./g) were associated with differences in the cardiac mitochondrial function 8 days post infection (p.i.). Infected C57BL/6 mouse hearts disclosed increased complex I (CI) and CIII activity, but restricted CII and normal CIV activity of RC. Reduced expression of the antioxidative catalase was accompanied by elevated lipid peroxidation (LPO), indicating oxidative stress. Intrinsic apoptosis was activated demonstrated by elevated levels of Bax, Bcl-2, caspase 3 and DNA degradation. In contrast, all myocardial RC complex activities were restricted in CVB3-infected A.SW/SnJ mice. The antioxidative system provided sufficient protection against oxidative stress shown by an elevated catalase expression and unaltered LPO. Bax and Bcl-2 levels were unchanged in CVB3-infected A.SW/SnJ mice, while caspase 3 was moderately increased but no DNA degradation was detectable. Correlation analyses including data from the two mouse strains revealed that reduced CVB3 titer correlated with increased CI and CIII activity, oxidative stress as well as active apoptosis during acute myocarditis (MC). C57BL/6 mice completely eliminated CVB3 and inflammation and normalized all intracellular parameters, while A.SW/SnJ mice showed permanently restricted CI activity in chronic MC 90 days p.i., at which time the replicating virus was no longer detectable but immunological processes were still active. Consequently, the regulation of energy metabolism appears crucial for an effective virus elimination and may be of prognostic and therapeutic significance for patients with virus-induced MC.
- Published
- 2012
- Full Text
- View/download PDF
33. Virus-host coevolution in a persistently coxsackievirus B3-infected cardiomyocyte cell line.
- Author
-
Pinkert S, Klingel K, Lindig V, Dörner A, Zeichhardt H, Spiller OB, and Fechner H
- Subjects
- Animals, Cell Survival, Cells, Cultured, Coxsackievirus Infections pathology, Coxsackievirus Infections virology, Enterovirus B, Human pathogenicity, Humans, Male, Mice, Mice, Inbred BALB C, Rats, Virulence, Biological Evolution, Enterovirus B, Human genetics, Enterovirus B, Human growth & development, Myocytes, Cardiac virology
- Abstract
Coevolution of virus and host is a process that emerges in persistent virus infections. Here we studied the coevolutionary development of coxsackievirus B3 (CVB3) and cardiac myocytes representing the major target cells of CVB3 in the heart in a newly established persistently CVB3-infected murine cardiac myocyte cell line, HL-1(CVB3). CVB3 persistence in HL-1(CVB3) cells represented a typical carrier-state infection with high levels (10(6) to 10(8) PFU/ml) of infectious virus produced from only a small proportion (approximately 10%) of infected cells. CVB3 persistence was characterized by the evolution of a CVB3 variant (CVB3-HL1) that displayed strongly increased cytotoxicity in the naive HL-1 cell line and showed increased replication rates in cultured primary cardiac myocytes of mouse, rat, and naive HL-1 cells in vitro, whereas it was unable to establish murine cardiac infection in vivo. Resistance of HL-1(CVB3) cells to CVB3-HL1 was associated with reduction of coxsackievirus and adenovirus receptor (CAR) expression. Decreasing host cell CAR expression was partially overcome by the CVB3-HL1 variant through CAR-independent entry into resistant cells. Moreover, CVB3-HL1 conserved the ability to infect cells via CAR. The employment of a soluble CAR variant resulted in the complete cure of HL-1(CVB3) cells with respect to the adapted virus. In conclusion, this is the first report of a CVB3 carrier-state infection in a cardiomyocyte cell line, revealing natural coevolution of CAR downregulation with CAR-independent viral entry in resistant host cells as an important mechanism of induction of CVB3 persistence.
- Published
- 2011
- Full Text
- View/download PDF
34. Transactivation of human parvovirus B19 gene expression in endothelial cells by adenoviral helper functions.
- Author
-
Pozzuto T, von Kietzell K, Bock T, Schmidt-Lucke C, Poller W, Zobel T, Lassner D, Zeichhardt H, Weger S, and Fechner H
- Subjects
- Adenovirus E1A Proteins metabolism, Adenovirus E4 Proteins metabolism, Cells, Cultured, Humans, Parvovirus B19, Human genetics, Transcription, Genetic, Adenoviruses, Human genetics, Endothelial Cells virology, Gene Expression Regulation, Viral, Parvovirus B19, Human physiology, Trans-Activators metabolism, Transcriptional Activation, Viral Proteins metabolism
- Abstract
Human parvovirus B19 (B19V) DNA is highly prevalent in endothelial cells lining up intramyocardial arterioles and postcapillary venules of patients with chronic myocarditis and cardiomyopathies. We addressed the question of a possible stimulation of B19V gene expression in endothelial cells by infection with adenoviruses. Adenovirus infection led to a strong augmentation of B19V structural and nonstructural proteins in individual endothelial cells infected with B19V or transfected with an infectious B19V genome. Transactivation was mostly mediated at the level of transcription and not due to adenovirus-mediated induction of second-strand synthesis from the single-stranded parvoviral genome. The main adenoviral functions required were E1A and E4orf6, which displayed synergistic effects. Furthermore, a limited B19V genome replication could be demonstrated in endothelial cells and adenovirus infection induced the appearance of putative dimeric replication intermediates. Thus the almost complete block in B19V gene expression seen in endothelial cells can be abrogated by infection with other viruses., (Copyright © 2010 Elsevier Inc. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
35. Application of small interfering RNAs modified by unlocked nucleic acid (UNA) to inhibit the heart-pathogenic coxsackievirus B3.
- Author
-
Werk D, Wengel J, Wengel SL, Grunert HP, Zeichhardt H, and Kurreck J
- Subjects
- Animals, Chlorocebus aethiops, Enterovirus B, Human genetics, RNA, Small Interfering genetics, Vero Cells, Antiviral Agents pharmacology, Enterovirus B, Human drug effects, Heart virology, Nucleic Acids chemistry, RNA, Small Interfering chemistry, RNA, Small Interfering pharmacology
- Abstract
This study describes the first application of unlocked nucleic acid (UNA)-modified small interfering RNAs (siRNAs) directed against a medically relevant target, the coxsackievirus B3. We systematically analyzed the impact of different siRNA modification patterns and observed good compatibility of the introduction of UNA with the maintenance of high antiviral activity. Additionally, the polarity of an siRNA was successfully reversed by modulating the relative stability of the termini with locked nucleic acid (LNA) and UNA as shown in a reporter assay. The potency of the reversed siRNA against the full-length target was, however, too low to inhibit the infectious virus. Altogether, combined modification of siRNAs with LNA und UNA provides a promising approach to alter and improve properties of an siRNA., (2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
36. Design of LNA-modified siRNAs against the highly structured 5' UTR of coxsackievirus B3.
- Author
-
Dutkiewicz M, Grunert HP, Zeichhardt H, Lena SW, Wengel J, and Kurreck J
- Subjects
- 5' Untranslated Regions genetics, Animals, Antiviral Agents chemistry, Cell Survival drug effects, Cell Survival genetics, Enterovirus B, Human genetics, Enterovirus B, Human physiology, HeLa Cells, Humans, Oligonucleotides chemistry, Oligonucleotides pharmacology, RNA, Small Interfering chemistry, RNA, Small Interfering pharmacology, Virus Replication drug effects, Virus Replication genetics, 5' Untranslated Regions antagonists & inhibitors, Antiviral Agents pharmacology, Enterovirus B, Human drug effects, Oligonucleotides genetics, RNA Interference, RNA, Small Interfering genetics
- Abstract
This study describes a strategy to develop LNA-modified small interfering RNA (siRNAs) against the highly structured 5' UTR of coxsackievirus B3 (CVB-3), which is an attractive target site due to its high degree of conservation. Accessible sites were identified based on structural models and RNase H assays with DNA oligonucleotides. Subsequently, LNA gapmers, siRNAs, siLNAs and small internally segmented interfering RNA (sisiLNAs) were designed against sites, which were found to be accessible in the in vitro assays, and tested in reporter assays and experiments with the infectious virus. The best siLNA improved viability of infected cells by 92% and exerted good antiviral activity in plaque reduction assays.
- Published
- 2008
- Full Text
- View/download PDF
37. Cardiac-targeted RNA interference mediated by an AAV9 vector improves cardiac function in coxsackievirus B3 cardiomyopathy.
- Author
-
Fechner H, Sipo I, Westermann D, Pinkert S, Wang X, Suckau L, Kurreck J, Zeichhardt H, Müller O, Vetter R, Erdmann V, Tschope C, and Poller W
- Subjects
- Adult, Animals, Base Sequence, Cardiomyopathies genetics, Cell Line, Child, Dependovirus genetics, Dependovirus metabolism, Genetic Vectors, Hemodynamics, Humans, Mice, Molecular Sequence Data, Myocardium cytology, RNA, Viral, RNA-Dependent RNA Polymerase genetics, RNA-Dependent RNA Polymerase metabolism, Rats, Viral Proteins genetics, Viral Proteins metabolism, Cardiomyopathies therapy, Cardiomyopathies virology, Coxsackievirus Infections therapy, Enterovirus B, Human enzymology, Enterovirus B, Human genetics, Heart physiology, Myocardium metabolism, RNA Interference
- Abstract
RNA interference (RNAi) has potential to be a novel therapeutic strategy in diverse areas of medicine. In this paper, we report on targeted RNAi for the treatment of a viral cardiomyopathy, which is a major cause of sudden cardiac death or terminal heart failure in children and young adults. RNAi therapy employs small regulatory RNAs to achieve its effect, but in vivo use of synthetic small interfering RNAs is limited by instability in plasma and low transfer into target cells. We instead evaluated an RNAi strategy using short hairpin RNA (shRdRp) directed at the RNA polymerase (RdRP) of coxsackievirus B3 (CoxB3) in HeLa cells, primary rat cardiomyocytes (PNCMs) and CoxB3-infected mice in vivo. A conventional AAV2 vector expressing shRdRp protected HeLa against virus-induced death, but this vector type was unable to transduce PNCMs. In contrast, an analogous pseudotyped AAV2.6 vector was protective also in PNCMs and reduced virus replication by >3 log10 steps. Finally, we evaluated the intravenous treatment of mice with an AAV2.9-shRdRp vector because AAV9 carries the most cardiotropic AAV capsid currently known for in vivo use. Mice with CoxB3 cardiomyopathy had disturbed left ventricular (LV) function with impaired parameters of contractility (dP/dtmax = 3,006 +/- 287 vs. 7,482 +/- 487 mmHg/s, p < 0.01) and diastolic relaxation (dP/dtmin = -2,224 +/- 195 vs. -6,456 +/- 356 mmHg/s, p < 0.01 and Tau = 16.2 +/- 1.1 vs. 10.7 +/- 0.6 ms, p < 0.01) compared to control mice. AAV2.9-shRdRp treatment significantly attenuated the cardiac dysfunction compared to control vector-treated mice on day 10 after CoxB3 infection: dP/dtmax = 3,865 +/- 354 vs. 3,006 +/- 287 mmHg/s (p < 0.05), dP/dtmin = -3,245 +/- 231 vs. -2,224 +/- 195 mmHg/s (p < 0.05) and Tau = 11.9 +/- 0.5 vs. 16.2 +/- 1.1 ms (p < 0.01). The data show, for the first time, that intravenously injected AAV9 has the potential to target RNAi to the heart and suggest AAV9-shRNA vectors as a novel therapeutic approach for cardiac disorders.
- Published
- 2008
- Full Text
- View/download PDF
38. Carvedilol improves left ventricular function in murine coxsackievirus-induced acute myocarditis association with reduced myocardial interleukin-1beta and MMP-8 expression and a modulated immune response.
- Author
-
Pauschinger M, Rutschow S, Chandrasekharan K, Westermann D, Weitz A, Peter Schwimmbeck L, Zeichhardt H, Poller W, Noutsias M, Li J, Schultheiss HP, and Tschope C
- Subjects
- Animals, Carvedilol, Coxsackievirus Infections physiopathology, Male, Matrix Metalloproteinases metabolism, Metoprolol pharmacology, Mice, Mice, Inbred BALB C, Myocarditis immunology, Myocarditis virology, RNA, Messenger, Tissue Inhibitor of Metalloproteinases metabolism, Adrenergic beta-Antagonists pharmacology, Carbazoles pharmacology, Interleukin-1 metabolism, Matrix Metalloproteinase 8 metabolism, Myocarditis physiopathology, Propanolamines pharmacology, Ventricular Function, Left drug effects
- Abstract
Background: Proinflammatory cytokines induce the expression of matrix metalloproteinases that play a crucial role in myocardial remodeling. Beta-adrenergic receptor stimulation influences the production of cytokines heralding the possibility of modulating cytokine production by beta-adrenergic blockers., Methods and Results: In a coxsackievirus B3 murine myocarditis model (BALB/c), effects of carvedilol and metoprolol on myocardial cytokine expression, inflammatory cell infiltration and MMP/TIMP profiles were investigated. In carvedilol-treated mice, a significant improvement in left ventricular function was documented 10 days post infection. In infected mice (n=10), IL-1beta, TNF-alpha, TGF-beta(1) and IL-10 myocardial mRNA abundance were increased significantly (240%, 200%, 161%, and 230%) compared to controls (n=10), while IL-15 mRNA was markedly reduced (70%). Infected mice showed significantly increased infiltrations with CD3-, CD4- and CD8-T-lymphocytes (730%, 1110%, 380%). In the infected mice, myocardial MMP/TIMP profiles presented a significant upregulation of membrane type-1 MMP, MMP-9, MMP-8 and MMP-3 (150%, 160%, 340%, and 270%) and a significant decrease in TIMP-4 levels (75%). Carvedilol attenuated over-expression of myocardial TGF-beta(1), IL-1beta and MMP-8 mRNA expression significantly and induced a relevant IL-10 mRNA expression in the infected mice (n=10). By an unchanged infiltration with CD3-T-lymphocytes, carvedilol showed a representative reduction in CD4-T-lymphocytes., Conclusion: Carvedilol treatment in experimental myocarditis leads to reduced expression of proinflammatory cytokines and MMPs, which contributes to reduced matrix degradation and ultimately to improved structural integrity of the heart. Besides the antiadrenergic potential, carvedilol is beneficial due to a wide range of biological activities (antiinflammatory, antifibrotic, antioxidative and immunomodulatory).
- Published
- 2005
- Full Text
- View/download PDF
39. RNA cleaving '10-23' DNAzymes with enhanced stability and activity.
- Author
-
Schubert S, Gül DC, Grunert HP, Zeichhardt H, Erdmann VA, and Kurreck J
- Subjects
- Binding Sites genetics, Catalytic Domain genetics, DNA, Catalytic chemistry, DNA, Catalytic genetics, DNA, Single-Stranded chemistry, DNA, Single-Stranded genetics, Enzyme Stability, Humans, Kinetics, Mutation, Nucleic Acid Denaturation, Oligoribonucleotides genetics, Oligoribonucleotides metabolism, RNA, Viral genetics, RNA, Viral metabolism, Rhinovirus genetics, Single-Strand Specific DNA and RNA Endonucleases metabolism, Substrate Specificity, Temperature, DNA, Catalytic metabolism, DNA, Single-Stranded metabolism
- Abstract
'10-23' DNAzymes can be used to cleave any target RNA in a sequence-specific manner. For applications in vivo, they have to be stabilised against nucleolytic attack by the introduction of modified nucleotides without obstructing cleavage activity. In this study, we optimise the design of a DNAzyme targeting the 5'-non-translated region of the human rhinovirus 14, a common cold virus, with regard to its kinetic properties and its stability against nucleases. We compare a large number of DNAzymes against the same target site that are stabilised by the use of a 3'-3'-inverted thymidine, phosphorothioate linkages, 2'-O-methyl RNA and locked nucleic acids, respectively. Both cleavage activity and nuclease stability were significantly enhanced by optimisation of arm length and content of modified nucleotides. Furthermore, we introduced modified nucleotides into the catalytic core to enhance stability against endonucleolytic degradation without abolishing catalytic activity. Our findings enabled us to establish a design for DNAzymes containing nucleotide modifications both in the binding arms and in the catalytic core, yielding a species with up to 10-fold enhanced activity and significantly elevated stability against nucleolytic cleavage. When transferring the design to a DNAzyme against a different target, only a slight modification was necessary to retain activity.
- Published
- 2003
- Full Text
- View/download PDF
40. Collagen degradation in a murine myocarditis model: relevance of matrix metalloproteinase in association with inflammatory induction.
- Author
-
Li J, Schwimmbeck PL, Tschope C, Leschka S, Husmann L, Rutschow S, Reichenbach F, Noutsias M, Kobalz U, Poller W, Spillmann F, Zeichhardt H, Schultheiss HP, and Pauschinger M
- Subjects
- Animals, Blotting, Western, Coxsackievirus Infections metabolism, Cytokines biosynthesis, Cytokines genetics, Disease Models, Animal, Down-Regulation, Hemodynamics, Immunoenzyme Techniques, Male, Matrix Metalloproteinases biosynthesis, Matrix Metalloproteinases genetics, Mice, Mice, Inbred BALB C, Myocarditis physiopathology, Myocarditis virology, RNA, Messenger genetics, Reverse Transcriptase Polymerase Chain Reaction, Tissue Inhibitor of Metalloproteinases biosynthesis, Tissue Inhibitor of Metalloproteinases genetics, Up-Regulation, Collagen metabolism, Matrix Metalloproteinases physiology, Myocarditis metabolism
- Abstract
Objective: Myocardial collagen degradation is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinase (TIMPs). The possible relevance of MMPs in association with the inflammatory induction was investigated in a murine coxsackievirus B3 myocarditis model., Methods: Hearts from viral infected and sham-infected BALB/c mice were analyzed by semi-quantitative RT-PCR, picrosirius red staining, Western blot analysis, and immunohistochemistry., Results: In viral infected mice, both mRNA and protein abundance for collagen type I remained unaltered. In addition, picrosirius red staining showed the unchanged total collagen content. However, degraded soluble fraction of collagen type I protein was increased. Moreover, the mRNA abundance for MMP-3 and MMP-9 was upregulated, whereas the mRNAs for TIMP-1 and TIMP-4 were downregulated, respectively. The upregulation of MMP-3/MMP-9 and downregulation of TIMP-4 were confirmed at the protein level, and were associated with significantly increased mRNA levels of interleukin 1beta, tumor necrosis factor-alpha, transforming growth factor-beta1 and interleukin-4., Conclusion: The increment of MMPs in the absence of counterbalance by TIMPs may lead to a functional defect of the myocardial collagen network by posttranslational mechanisms. This may contribute significantly to the development of left ventricular dysfunction in murine viral myocarditis. The inflammatory response with induction of cytokines may mediate the dysregulation of the myocardial MMP/TIMP systems.
- Published
- 2002
- Full Text
- View/download PDF
41. Detection of antibodies against viral capsid proteins of human herpesvirus 8 in AIDS-associated Kaposi's sarcoma.
- Author
-
André S, Schatz O, Bogner JR, Zeichhardt H, Stöffler-Meilicke M, Jahn HU, Ullrich R, Sonntag AK, Kehm R, and Haas J
- Subjects
- Amino Acid Sequence, Antibodies, Viral analysis, Humans, Molecular Sequence Data, Sequence Alignment, Sequence Homology, Amino Acid, Capsid immunology, Herpesvirus 8, Human immunology, Sarcoma, Kaposi immunology
- Abstract
Sequences of a new herpesvirus with homology to gammaherpesvirinae were recently identified in AIDS-associated Kaposi's sarcoma (KS). Subsequently this novel virus, called KS-associated virus (KSHV) or human herpesvirus (HHV) 8 was detected in classical KS and AIDS-associated body cavity based lymphomas by polymerase chain reaction. In this report major and minor capsid proteins of HHV-8 were molecularly cloned and produced as recombinant proteins in Escherichia coli. Sera from 69 HIV-1 infected patients with KS, 30 HIV-1 infected patients without KS and 106 control individuals were tested by enzyme-linked immunosorbent assay for anti-HHV-8 capsid IgM and IgG antibodies. Sera from four patients were tested over periods ranging from 18 months to 6 years. IgG antibodies directed against HHV-8 capsid antigens were detected in patients with AIDS-associated KS and in some AIDS patients without KS. Seroconversion with IgM and IgG antibodies directed against HHV-8 capsid proteins occurred more than 1 year prior to diagnosis of KS. In a considerable portion of KS patients no IgM or IgG antibodies against HHV-8 capsid proteins were detected. In these patients there was an inverse relationship between antibodies against HHV-8orf26 and the CD4/CD8 ratio, suggesting that the inconsistency of anti-HHV-8orf26 antibodies is due at least partly to an impaired immune response. No reactivity against HHV-8 capsid antigens was detected in the vast majority of sera from HIV-negative control individuals. Our findings indicate that a specific humoral immune response against capsid proteins is raised in HHV-8 infected individuals, and that anti-capsid antibodies can be used to diagnose HHV-8 infection. The correlation between occurrence of anti-HHV-8 antibodies and KS supports the hypothesis of a causative role of HHV-8.
- Published
- 1997
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.