79 results on '"Gosert R"'
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2. Membrane association of hepatitis C virus nonstructural proteins and identification of the membrane alteration that harbors the viral replication complex
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Moradpour, D., Gosert, R., Egger, D., Penin, F., Blum, He, Bienz, K., and Deleage, Gilbert
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[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,virus diseases - Abstract
Hepatitis C virus (HCV) replicates its genome in a membrane-associated complex composed of viral proteins, replicating RNA, and altered cellular membranes. Determinants for membrane association of the HCV nonstructural proteins involved in genome replication have been defined. In addition, a specific membrane alteration, designated membranous web, was recently identified as the site of viral RNA synthesis and, therefore, represents the HCV replication complex. These findings add to our current understanding of the HCV life cycle and may ultimately allow to design novel antiviral strategies against hepatitis C.Hepatitis C virus (HCV) replicates its genome in a membrane-associated complex composed of viral proteins, replicating RNA, and altered cellular membranes. Determinants for membrane association of the HCV nonstructural proteins involved in genome replication have been defined. In addition, a specific membrane alteration, designated membranous web, was recently identified as the site of viral RNA synthesis and, therefore, represents the HCV replication complex. These findings add to our current understanding of the HCV life cycle and may ultimately allow to design novel antiviral strategies against hepatitis C.
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- 2003
3. Transient Expression of Cellular Polypyrimidine-Tract Binding Protein Stimulates Cap-Independent Translation Directed by Both Picornaviral and Flaviviral Internal Ribosome Entry Sites In Vivo
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Sangar, D. V., Chang, K. H., Lemon, S. M., Gosert, R., Rijnbrand, R., and Yi, M.
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integumentary system ,macromolecular substances ,environment and public health - Abstract
The regulation of cap-independent translation directed by the internal ribosome entry sites (IRESs) present in some viral and cellular RNAs is poorly understood. Polypyrimidine-tract binding protein (PTB) binds specifically to several viral IRESs. IRES-directed translation may be reduced in cell-free systems that are depleted of PTB and restored by reconstitution of lysates with recombinant PTB. However, there are no data concerning the effects of PTB on IRES-directed translation in vivo. We transfected cells with plasmids expressing dicistronic transcripts in which the upstream cistron encoded PTB or PTB deletion mutants (including a null mutant lacking amino acid residues 87 to 531). The downstream cistron encoded a reporter protein (chloramphenicol acetyltransferase [CAT]) under translational control of the poliovirus IRES which was placed within the intercistronic space. In transfected BS-C-1 cells, transcripts expressing wild-type PTB produced 12-fold more reporter protein than similar transcripts encoding the PTB null mutant. There was a 2.4-fold difference in CAT produced from these transcripts in HeLa cells, which contain a greater natural abundance of PTB. PTB similarly stimulated CAT production from transcripts containing the IRES of hepatitis A virus or hepatitis C virus in BS-C-1 cells and Huh-7 cells (37- to 44-fold increase and 5 to 5.3-fold increase, respectively). Since PTB had no quantitative or qualitative effect on transcription from these plasmids, we conclude that PTB stimulates translation of representative picornaviral and flaviviral RNAs in vivo. This is likely to reflect the stabilization of higher ordered RNA structures within the IRES and was not observed with PTB mutants lacking RNA recognition motifs located in the C-terminal third of the molecule.
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- 2000
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4. Management and outcome of CSF-JC virus PCR-negative PML in a natalizumab-treated patient with MS.
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Kuhle J, Gosert R, Bühler R, Derfuss T, Sutter R, Yaldizli O, Radue EW, Ryschkewitsch C, Major EO, Kappos L, Frank S, Hirsch HH, Kuhle, J, Gosert, R, Bühler, R, Derfuss, T, Sutter, R, Yaldizli, O, Radue, E-W, and Ryschkewitsch, C
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- 2011
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5. OP4-6 The polyomavirus BK agnoprotein co-localizes with lipid droplets
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Unterstab, G., primary, Gosert, R., additional, Leuenberger, D., additional, Lorentz, P., additional, Rinaldo, C.H., additional, and Hirsch, H.H., additional
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- 2009
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6. OP5-8 Hexadecyloxypropyl-cidofovir (CMX001) BK virus (BKV) replication in primary renal tubular epithelial cells
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Rinaldo, C.H., primary, Gosert, R., additional, and Hirsch, H.H., additional
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- 2009
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7. OP4-7 Polyomavirus JC (JCV) with rearranged noncoding control region are found in cerebrospinal fluid, but not in urine and increase viral early gene expression
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Gosert, R., primary, Kardas, P., additional, Major, E.O., additional, and Hirsch, H.H., additional
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- 2009
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8. Cytomegalovirus and polyomavirus BK posttransplant
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Egli, A., primary, Binggeli, S., additional, Bodaghi, S., additional, Dumoulin, A., additional, Funk, G. A., additional, Khanna, N., additional, Leuenberger, D., additional, Gosert, R., additional, and Hirsch, H. H., additional
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- 2007
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9. Membrane association of hepatitis C virus nonstructural proteins and identification of the membrane alteration that harbors the viral replication complex
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MORADPOUR, D, primary, GOSERT, R, additional, EGGER, D, additional, PENIN, F, additional, BLUM, H, additional, and BIENZ, K, additional
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- 2003
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10. Identification of active-site residues in protease 3C of hepatitis A virus by site-directed mutagenesis
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Gosert, R, primary, Dollenmaier, G, additional, and Weitz, M, additional
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- 1997
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11. Identification of hepatitis A virus non-structural protein 2B and its release by the major virus protease 3C
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Gosert, R., primary, Cassinotti, P., additional, Siegl, G., additional, and Weitz, M., additional
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- 1996
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12. Polyomavirus BK Replication Dynamics In Vivoand In Silicoto Predict Cytopathology and Viral Clearance in Kidney Transplants
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Funk, G. A., Gosert, R., Comoli, P., Ginevri, F., and Hirsch, H. H.
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Fast BK virus (BKV) replication in renal tubular epithelial cells drives polyomavirus-BK-associated nephropathy (PVAN) to premature kidney transplant (KT) failure. BKV also replicates in urothelial cells, but remains asymptomatic in two-thirds of affected KT patients. Comparing 518 day-matched plasma-urine samples from 223 KT patients, BKV loads were ?3000-fold higher in urine than in plasma (p < 0.000001). Molecular and quantitative parameters indicated that >95 of urine BKV loads resulted from urothelial replication and <5 from tubular epithelial replication. Fast BKV replication dynamics in plasma and urine with half-lives of <12 h accounted for daily urothelial and tubular epithelial cell loss of 4×107and 6×107, respectively. BKV dynamics in both sites were only partly linked, with full and partial discordance in 36 and 32, respectively. Viral expansion was best explained by models where BKV replication started in the kidney followed by urothelial amplification and tubular epithelial cell cross-feeding reaching a dynamic equilibrium after ?10 weeks. Curtailing intrarenal replication by 50 was ineffective and >80 was required for clearing viremia within 7 weeks, but viruria persisted for >14 weeks. Reductions >90 cleared viremia and viruria by 3 and 10 weeks, respectively. The model was clinically validated in prospectively monitored KT patients supporting >80 curtailing for optimal interventions.
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- 2008
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13. Steatosis and liver cancer in transgenic mice expressing the structural and nonstructural proteins of hepatitis c virus
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Lerat, H., Honda, M., Beard, M.R., Loesch, K., Sun, J., Yang, Y., Okuda, M., Gosert, R., Xiao, S.Y., Weinman, S.A., and Lemon, S.M.
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Background & Aims:The aim of this study was to determine whether expression of hepatitis C virus proteins alters hepatic morphology or function in the absence of inflammation. Methods:Transgenic C57BL/6 mice with liver-specific expression of RNA encoding the complete viral polyprotein (FL-N transgene) or viral structural proteins (S-N transgene) were compared with nontransgenic littermates for altered liver morphology and function. Results:FL-N transcripts were detectable only by reverse-transcription polymerase chain reaction, and S-N transcripts were identified in Northern blots. The abundance of viral proteins was sufficient for detection only in S-N transgenic animals. There was no inflammation in transgenic livers, but mice expressing either transgene developed age-related hepatic steatosis that was more severe in males. Apoptotic or proliferating hepatocytes were not significantly increased. Hepatocellular adenoma or carcinoma developed in older male animals expressing either transgene, but their incidence reached statistical significance only in FL-N animals. Neither was ever observed in age-matched nontransgenic mice. Conclusions:Constitutive expression of viral proteins leads to common pathologic features of hepatitis C in the absence of specific anti-viral immune responses. Expression of the structural proteins enhances a low background of steatosis in C57BL/6 mice, while additional low level expression of nonstructural proteins increases the risk of cancer.
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- 2002
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14. Characterization of highly frequent epitope-specific CD45RA+/CCR7+/- T lymphocyte responses against p53-binding domains of the human polyomavirus BK large tumor antigen in HLA-A*0201+ BKV-seropositive donors
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Zajac Paul, Gosert Rainer, Sais Giovanni, Hudolin Tvrtko, Wyler Stephen, Bracci Laura, Provenzano Maurizio, Palu' Giorgio, Heberer Michael, Hirsch Hans H, and Spagnoli Giulio C
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Medicine - Abstract
Abstract Human polyomavirus BK (BKV) has been implicated in oncogenic transformation. Its ability to replicate is determined by the binding of its large tumor antigen (LTag) to products of tumor-suppressor genes regulating cell cycle, as specifically p53. We investigated CD8+ T immune responses to BKV LTag portions involved in p53 binding in HLA-A*0201+ BKV LTag experienced individuals. Peptides selected from either p53-binding region (LTag351–450 and LTag533–626) by current algorithms and capacity to bind HLA-A*0201 molecule were used to stimulate CD8+ T responses, as assessed by IFN-γ gene expression ex vivo and detected by cytotoxicity assays following in vitro culture. We observed epitope-specific immune responses in all HLA-A*0201+ BKV LTag experienced individuals tested. At least one epitope, LTag579–587; LLLIWFRPV, was naturally processed in non professional antigen presenting cells and induced cytotoxic responses with CTL precursor frequencies in the order of 1/20'000. Antigen specific CD8+ T cells were only detectable in the CD45RA+ subset, in both CCR7+ and CCR7- subpopulations. These data indicate that widespread cellular immune responses against epitopes within BKV LTag-p53 binding regions exist and question their roles in immunosurveillance against tumors possibly associated with BKV infection.
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- 2006
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15. Revealing the unseen: next generation sequencing for early detection of drug-resistant cytomegalovirus variants upon letermovir prophylaxis failure.
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Nägele K, Bättig V, Gosert R, Walti CS, Prince SS, Halter J, Mathews R, Stühler C, Khanna N, and Leuzinger K
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In allogeneic hematopoietic cell transplant (HCT)-recipients, prophylactic management strategies are essential for preventing CMV-reactivation and associated disease. We report on a 63-year-old male patient with a D-/R+ CMV-serostatus, who showed ongoing low-level CMV-replication post-HCT despite receiving letermovir prophylaxis. Sanger-sequencing failed to detect drug resistance mutations (DRM) until CMV-pneumonitis developed, revealing a UL56-C325R-DRM linked to high-level letermovir resistance. Retrospective analysis with next-generation-sequencing (NGS) revealed the DRM at a low frequency of 6% two weeks prior to detection by Sanger-sequencing. This study highlights the importance of advanced NGS-methods for early detection of CMV-DRMs, allowing for faster adjustments in antiviral treatment strategies., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)
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- 2024
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16. Impact of nucleic acid extraction procedures on human papillomavirus (HPV) detection and genotyping.
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Naegele K, Weissbach FH, Leuzinger K, Gosert R, Bubendorf L, and Hirsch HH
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- Female, Humans, Human Papillomavirus Viruses, Genotype, Papillomaviridae genetics, DNA, Viral genetics, Papillomavirus Infections, Uterine Cervical Neoplasms, Precancerous Conditions, Carcinoma, Squamous Cell
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Human papillomavirus (HPV) infections are often asymptomatic, but some of the >200 HPV genotypes confer a high risk for precancerous cervical lesions and cervical cancer. Current clinical management of HPV infections relies on reliable nucleic acid testing detection and genotyping. We prospectively compared nucleic acid extraction without and with prior centrifugation enrichment for detecting and genotyping HPV in cervical swabs with atypical squamous or glandular cells. Consecutive swabs were analyzed from 45 patients with atypical squamous or glandular cells. Nucleic acids were extracted in parallel using three procedures, Abbott-M2000, Roche-MagNA-Pure-96 Large-Volume Kit without (Roche-MP-large) and with prior centrifugation (Roche-MP-large/spin) and tested using Seegene-Anyplex-II HPV28. In total, 54 HPV-genotypes were detected in 45 samples, 51 by Roche-MP-large/spin, 48 by Abbott-M2000 and 42 by Roche-MP-large. The overall concordance was 80% for detecting any HPV and 74% for specific HPV-genotypes. Roche-MP-large/spin and Abbott-M2000 showed the highest concordance for HPV detection (88.9%; kappa 0.78), and genotyping (88.5%). Two and more HPV-genotypes were detected in 15 samples, often with one HPV being more abundant. Dilution series confirmed the specific detection of multiple HPV-genotypes and their relative abundance. In 285 consecutive follow-up samples extracted by Roche-MP-large/spin, the top three detected genotypes were the high-risk HPV16, HPV53, HPV56 and the low-risk HPV42, HPV54 and HPV61. Rate and breadth of HPV detection in cervical swabs depends on extraction protocols being highest after centrifugation/enrichment. As multivalent HPV-vaccine coverage increases, detecting the evolving HPV virome depends on improved extraction and broader genotype coverage., (© 2023 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)
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- 2023
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17. Usefulness of the GenMark ePlex RPP assay for the detection of respiratory viruses compared to the FTD21 multiplex RT-PCR.
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Steiner F, Schmutz S, Gosert R, Huder JB, Redli PM, Capaul R, Hirsch HH, Böni J, and Zbinden A
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- Diagnostic Tests, Routine, Humans, Respiratory Tract Infections virology, Sensitivity and Specificity, Time Factors, Viruses classification, Viruses genetics, Molecular Diagnostic Techniques, Multiplex Polymerase Chain Reaction, Respiratory Tract Infections diagnosis, Viruses isolation & purification
- Abstract
Cartridge-based multiplex panels covering numerous pathogens offer an advantage of minimal hands-on-time and short time to result to commercial RT-PCR assays. In this study, we evaluated the performance of the ePlex respiratory pathogen panel (RPP) compared to the Fast Track Diagnostics Respiratory pathogens 21 multiplex RT-PCR assay (FTD21) using 400 clinical respiratory samples. Discrepant results were further analysed by a reference nucleic acid amplification testing (NAT) and a composite reference approach was used for final interpretation. Discordant results were observed in 56 targets corresponding to 54 samples. Sensitivities and specificities were 85.5% and 99.9% for the ePlex RPP and 95.8% and 99.7% for the FTD21 system, respectively. Altogether, the ePlex RPP is a valuable tool for the rapid detection of a number of different respiratory viruses with the exception of the coronavirus family (low sensitivity ranging from 50-80%) and samples with a low pathogen load (Ct values >33)., Competing Interests: Disclosures The authors declare no conflict of interest., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)
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- 2021
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18. Comparing cytomegalovirus diagnostics by cell culture and quantitative nucleic acid testing in broncho-alveolar lavage fluids.
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Leuzinger K, Stolz D, Gosert R, Naegele K, Prince SS, Tamm M, and Hirsch HH
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- Adult, Aged, Aged, 80 and over, Cell Culture Techniques methods, Cytomegalovirus Infections virology, DNA, Viral genetics, Female, Humans, Male, Middle Aged, Prospective Studies, Retrospective Studies, Virology methods, Young Adult, Bronchoalveolar Lavage Fluid virology, Cytomegalovirus genetics, Cytomegalovirus growth & development, Cytomegalovirus Infections diagnosis, Viral Load methods
- Abstract
Many clinical laboratories have replaced virus isolation in cell-culture (VIC) for cytomegalovirus (CMV) by quantitative-nucleic-acid testing (QNAT), rendering clinically relevant CMV-replication difficult to distinguish from CMV-shedding or latent infection. We compared direct VIC in 1109 consecutive bronchoalveolar lavage fluids (BALFs) and a well-validated CMV-QNAT (Basel-CMV-UL111a-77bp). In the retrospective Group 1 (N = 694) and Group 2 (N = 303), CMV-QNAT was performed within 48 h from 2-fold and 10-fold concentrated total nucleic acid (TNA) eluates, respectively. In Group 3 (N = 112), 2-fold and 10-fold concentrated TNA eluates were prospectively analyzed in parallel to VIC. CMV was detected by VIC in 79 of 694 (11%) and 26 of 303 (9%) of Groups 1 and 2, but in 114 of 694 (16%) and 57 of 303 (17%) by CMV-QNAT, respectively. Median CMV loads were significantly higher in VIC-positive than in VIC-negative BALF. The likelihood for CMV detection by VIC was 85% for BALF CMV- loads >4 log
10 copies/ml. In the prospective Group 3, CMV was detected by VIC in 10 of 112 (9%), and in 14 of 112 (13%) and 20 of 112 (18%) by CMV-QNAT, when using 2-fold and 10-fold concentrated TNA eluates, respectively. Notably, CMV was undetectable by CMV-QNAT in 10 VIC-positive cases of Groups 1 and 2, but in none of Group 3. We conclude that CMV-QNAT can be adopted to BALF diagnostics but requires several careful steps in validation. CMV-QNAT loads >10 000 copies/ml in BALF may indicate significant CMV replication as defined by VIC, if short shipment and processing procedures can be guaranteed. Discordance of detecting CMV in time-matched plasma samples emphasises the role of local pulmonary CMV replication, for which histopathology remains the gold standard of proven CMV pneumonia., (© 2020 Wiley Periodicals LLC.)- Published
- 2021
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19. Epidemiology and precision of SARS-CoV-2 detection following lockdown and relaxation measures.
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Leuzinger K, Gosert R, Søgaard KK, Naegele K, Bielicki J, Roloff T, Bingisser R, Nickel CH, Khanna N, Sutter ST, Widmer AF, Rentsch K, Pargger H, Siegemund M, Stolz D, Tamm M, Bassetti S, Osthoff M, Battegay M, Egli A, and Hirsch HH
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- Adult, Bronchoalveolar Lavage, COVID-19 prevention & control, COVID-19 transmission, COVID-19 virology, COVID-19 Testing, Disease Transmission, Infectious prevention & control, Female, Genome, Viral, Humans, Male, Middle Aged, Nasopharynx virology, Oropharynx virology, Pandemics, RNA, Viral analysis, RNA, Viral genetics, SARS-CoV-2 genetics, Switzerland epidemiology, Viral Load, COVID-19 epidemiology, Communicable Disease Control methods, SARS-CoV-2 isolation & purification
- Abstract
Objectives: Detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is key to the clinical and epidemiological assessment of CoVID-19. We cross-validated manual and automated high-throughput testing for SARS-CoV-2-RNA, evaluated SARS-CoV-2 loads in nasopharyngeal-oropharyngeal swabs (NOPS), lower respiratory fluids, and plasma, and analyzed detection rates after lockdown and relaxation measures., Methods: Basel-S-gene, Roche-E-gene, and Roche-cobas®6800-Target1 and Target2 were prospectively validated in 1344 NOPS submitted during the first pandemic peak (Week 13). Follow-up cohort (FUP) 1, 2, and 3 comprised 10,999, 10,147, and 19,389 NOPS submitted during a 10-week period until Weeks 23, 33, and 43, respectively., Results: Concordant results were obtained in 1308 cases (97%), including 97 (9%) SARS-CoV-2-positives showing high quantitative correlations (Spearman's r > .95; p < .001) for all assays and high precision by Bland-Altman analysis. Discordant samples (N = 36, 3%) had significantly lower SARS-CoV-2 loads (p < .001). Following lockdown, detection rates declined to <1% in FUP-1, reducing single-test positive predictive values from 99.3% to 85.1%. Following relaxation, rates flared up to 4% and 12% in FUP-2 and -3, but infected patients were younger than during lockdown (34 vs. 52 years, p < .001). In 261 patients providing 936 NOPS, SARS-CoV-2 loads declined by three orders of magnitude within 10 days postdiagnosis (p < .001). SARS-CoV-2 loads in NOPS correlated with those in time-matched lower respiratory fluids or in plasma but remained detectable in some cases with negative follow-up NOPS, respectively., Conclusion: Manual and automated assays significantly correlated qualitatively and quantitatively. Following a successful lockdown, declining positive predictive values require independent dual-target confirmation for reliable assessment. Confirmatory and quantitative follow-up testing should be obtained within <5 days and consider lower respiratory fluids in symptomatic patients with SARS-CoV-2-negative NOPS., (© 2020 Wiley Periodicals LLC.)
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- 2021
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20. Corrigendum to: Epidemiology of Severe Acute Respiratory Syndrome Coronavirus 2 Emergence Amidst Community-Acquired Respiratory Viruses.
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Leuzinger K, Roloff T, Gosert R, Sogaard K, Naegele K, Rentsch K, Bingisser R, Nickel CH, Pargger H, Bassetti S, Bielicki J, Khanna N, Tschudin Sutter S, Widmer A, Hinic V, Battegay M, Egli A, and Hirsch HH
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- 2021
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21. Hunting coronavirus by transmission electron microscopy - a guide to SARS-CoV-2-associated ultrastructural pathology in COVID-19 tissues.
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Hopfer H, Herzig MC, Gosert R, Menter T, Hench J, Tzankov A, Hirsch HH, and Miller SE
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- COVID-19 virology, Coronavirus Infections pathology, Coronavirus Infections virology, Humans, Microscopy, Electron, Transmission, RNA, Viral, SARS-CoV-2 physiology, Virion ultrastructure, Virus Assembly, Virus Replication, COVID-19 pathology, SARS-CoV-2 ultrastructure
- Abstract
Transmission electron microscopy has become a valuable tool to investigate tissues of COVID-19 patients because it allows visualisation of SARS-CoV-2, but the 'virus-like particles' described in several organs have been highly contested. Because most electron microscopists in pathology are not accustomed to analysing viral particles and subcellular structures, our review aims to discuss the ultrastructural changes associated with SARS-CoV-2 infection and COVID-19 with respect to pathology, virology and electron microscopy. Using micrographs from infected cell cultures and autopsy tissues, we show how coronavirus replication affects ultrastructure and put the morphological findings in the context of viral replication, which induces extensive remodelling of the intracellular membrane systems. Virions assemble by budding into the endoplasmic reticulum-Golgi intermediate complex and are characterised by electron-dense dots of cross-sections of the nucleocapsid inside the viral particles. Physiological mimickers such as multivesicular bodies or coated vesicles serve as perfect decoys. Compared to other in-situ techniques, transmission electron microscopy is the only method to visualise assembled virions in tissues, and will be required to prove SARS-CoV-2 replication outside the respiratory tract. In practice, documenting in tissues the characteristic features seen in infected cell cultures seems to be much more difficult than anticipated. In our view, the hunt for coronavirus by transmission electron microscopy is still on., (© 2020 John Wiley & Sons Ltd.)
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- 2021
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22. Brief validation of the novel GeneXpert Xpress SARS-CoV-2 PCR assay.
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Goldenberger D, Leuzinger K, Sogaard KK, Gosert R, Roloff T, Naegele K, Cuénod A, Mari A, Seth-Smith H, Rentsch K, Hinić V, Hirsch HH, and Egli A
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- Betacoronavirus genetics, COVID-19, COVID-19 Testing, Clinical Laboratory Techniques instrumentation, Clinical Laboratory Techniques standards, Coronavirus Infections diagnosis, Diagnostic Tests, Routine, Humans, Nasopharynx virology, Pandemics, Pneumonia, Viral diagnosis, Polymerase Chain Reaction instrumentation, Polymerase Chain Reaction methods, Reproducibility of Results, SARS-CoV-2, Sensitivity and Specificity, Betacoronavirus isolation & purification, Clinical Laboratory Techniques methods, Coronavirus Infections virology, Pneumonia, Viral virology
- Abstract
The clinical and epidemiologic management of the SARS-CoV-2 pandemic is critically dependent on molecular assays with short turn-around time. We validated the novel Xpert Xpress SARS-CoV-2 assay using a commercial nucleic acid testing (Roche Cobas 6800). We found an excellent concordance over a range of SARS-CoV-2 loads and across established human coronaviruses., (Copyright © 2020. Published by Elsevier B.V.)
- Published
- 2020
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23. Epidemiology of Severe Acute Respiratory Syndrome Coronavirus 2 Emergence Amidst Community-Acquired Respiratory Viruses.
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Leuzinger K, Roloff T, Gosert R, Sogaard K, Naegele K, Rentsch K, Bingisser R, Nickel CH, Pargger H, Bassetti S, Bielicki J, Khanna N, Tschudin Sutter S, Widmer A, Hinic V, Battegay M, Egli A, and Hirsch HH
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- Adolescent, Adult, Aged, Aged, 80 and over, Betacoronavirus genetics, Betacoronavirus isolation & purification, COVID-19, COVID-19 Testing, Child, Child, Preschool, Clinical Laboratory Techniques, Coinfection immunology, Coinfection virology, Communicable Diseases, Emerging virology, Coronavirus Envelope Proteins, Coronavirus Infections diagnosis, Coronavirus Infections virology, Coronavirus Nucleocapsid Proteins, Female, Humans, Infant, Infant, Newborn, Male, Middle Aged, Nucleocapsid Proteins genetics, Pandemics, Phosphoproteins, Pneumonia, Viral diagnosis, Pneumonia, Viral virology, Respiratory Tract Infections virology, SARS-CoV-2, Spike Glycoprotein, Coronavirus genetics, Viral Envelope Proteins, World Health Organization, Young Adult, Coinfection epidemiology, Communicable Diseases, Emerging epidemiology, Coronavirus Infections epidemiology, Pneumonia, Viral epidemiology, Respiratory Tract Infections epidemiology
- Abstract
Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China as the cause of coronavirus disease 2019 in December 2019 and reached Europe by late January 2020, when community-acquired respiratory viruses (CARVs) are at their annual peak. We validated the World Health Organization (WHO)-recommended SARS-CoV-2 assay and analyzed the epidemiology of SARS-CoV-2 and CARVs., Methods: Nasopharyngeal/oropharyngeal swabs (NOPS) from 7663 patients were prospectively tested by the Basel S-gene and WHO-based E-gene (Roche) assays in parallel using the Basel N-gene assay for confirmation. CARVs were prospectively tested in 2394 NOPS by multiplex nucleic acid testing, including 1816 (75%) simultaneously for SARS-CoV-2., Results: The Basel S-gene and Roche E-gene assays were concordant in 7475 cases (97.5%) including 825 (11%) SARS-CoV-2 positives. In 188 (2.5%) discordant cases, SARS-CoV-2 loads were significantly lower than in concordant positive ones and confirmed in 105 (1.4%). Adults were more frequently SARS-CoV-2 positive, whereas children tested more frequently CARV positive. CARV coinfections with SARS-CoV-2 occurred in 1.8%. SARS-CoV-2 replaced CARVs within 3 weeks, reaching 48% of all detected respiratory viruses followed by rhinovirus/enterovirus (13%), influenza virus (12%), coronavirus (9%), respiratory syncytial virus (6%), and metapneumovirus (6%)., Conclusions: Winter CARVs were dominant during the early SARS-CoV-2 pandemic, impacting infection control and treatment decisions, but were rapidly replaced, suggesting competitive infection. We hypothesize that preexisting immune memory and innate immune interference contribute to the different SARS-CoV-2 epidemiology among adults and children., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)
- Published
- 2020
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24. Comparing the Cobas Liat Influenza A/B and respiratory syncytial virus assay with multiplex nucleic acid testing.
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Gosert R, Naegele K, and Hirsch HH
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- Adolescent, Adult, Aged, Aged, 80 and over, Child, Child, Preschool, Female, Humans, Infant, Male, Middle Aged, Prospective Studies, Sensitivity and Specificity, Young Adult, Diagnostic Tests, Routine methods, Influenza A virus isolation & purification, Influenza B virus isolation & purification, Influenza, Human diagnosis, Respiratory Syncytial Virus Infections diagnosis, Respiratory Syncytial Virus, Human isolation & purification
- Abstract
Influenza virus and respiratory syncytial virus (RSV) detection with short turn-around-time (TAT) is pivotal for rapid decisions regarding treatment and infection control. However, negative rapid testing results may come from poor assay sensitivity or from influenza-like illnesses caused by other community-acquired respiratory viruses (CARVs). We prospectively compared the performance of Cobas Liat Influenza A/B and RSV assay (LIAT) with our routine multiplexNAT-1 (xTAG Respiratory Pathogen Panel; Luminex) and multiplexNAT-2 (ePlex-RPP; GenMark Diagnostics) using 194 consecutive nasopharyngeal swabs from patients with influenza-like illness during winter 2017/2018. Discordant results were reanalyzed by specific in-house quantitative nucleic acid amplification testing (NAT). LIAT was positive for influenza virus-A, -B, and RSV in 18 (9.3%), 13 (6.7%), and 55 (28.4%) samples, and negative in 108 samples. Other CARVs were detected by multiplexNAT in 66 (34.0%) samples. Concordant results for influenza and RSV were seen in 190 (97.9%), discordant results in 4 (2.1%), which showed low-level RSV (<40 000 copies/mL). Sensitivity and specificity of LIAT for influenza-A, -B, and RSV were 100%, 100% and 100%, and 100%, 99.5% and 100%, respectively. The average TAT of LIAT was 20 minutes compared to 6 hours and 2 hours for the multiplexNAT-1 and -2, respectively. Thus, LIAT demonstrated excellent sensitivity and specificity for influenza and RSV, which together with the simple sample processing and short TAT renders this assay suitable for near-patient testing., (© 2018 Wiley Periodicals, Inc.)
- Published
- 2019
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25. Cytomegalovirus sequence variability, amplicon length, and DNase-sensitive non-encapsidated genomes are obstacles to standardization and commutability of plasma viral load results.
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Naegele K, Lautenschlager I, Gosert R, Loginov R, Bir K, Helanterä I, Schaub S, Khanna N, and Hirsch HH
- Subjects
- Cytomegalovirus classification, Cytomegalovirus genetics, DNA, Viral blood, DNA, Viral chemistry, DNA-Directed DNA Polymerase genetics, Humans, Mutation, Plasma virology, Prospective Studies, Retrospective Studies, Sequence Analysis, DNA, United Kingdom, Viral Proteins genetics, Cytomegalovirus isolation & purification, Cytomegalovirus Infections virology, DNA, Viral genetics, Deoxyribonucleases blood, Genetic Variation, Viral Load methods, Viral Load standards
- Abstract
Background: Cytomegalovirus (CMV) management post-transplantation relies on quantification in blood, but inter-laboratory and inter-assay variability impairs commutability. An international multicenter study demonstrated that variability is mitigated by standardizing plasma volumes, automating DNA extraction and amplification, and calibration to the 1st-CMV-WHO-International-Standard as in the FDA-approved Roche-CAP/CTM-CMV. However, Roche-CAP/CTM-CMV showed under-quantification and false-negative results in a quality assurance program (UK-NEQAS-2014)., Objectives: To evaluate factors contributing to quantification variability of CMV viral load and to develop optimized CMV-UL54-QNAT., Study Design: The UL54 target of the UK-NEQAS-2014 variant was sequenced and compared to 329 available CMV GenBank sequences. Four Basel-CMV-UL54-QNAT assays of 361 bp, 254 bp, 151 bp, and 95 bp amplicons were developed that only differed in reverse primer positions. The assays were validated using plasmid dilutions, UK-NEQAS-2014 sample, as well as 107 frozen and 69 prospectively collected plasma samples from transplant patients submitted for CMV QNAT, with and without DNase-digestion prior to nucleic acid extraction., Results: Eight of 43 mutations were identified as relevant in the UK-NEQAS-2014 target. All Basel-CMV-UL54 QNATs quantified the UK-NEQAS-2014 but revealed 10-fold increasing CMV loads as amplicon size decreased. The inverse correlation of amplicon size and viral loads was confirmed using 1st-WHO-International-Standard and patient samples. DNase pre-treatment reduced plasma CMV loads by >90% indicating the presence of unprotected CMV genomic DNA., Conclusions: Sequence variability, amplicon length, and non-encapsidated genomes obstruct standardization and commutability of CMV loads needed to develop thresholds for clinical research and management. Besides regular sequence surveys, matrix and extraction standardization, we propose developing reference calibrators using 100 bp amplicons., (Copyright © 2018 Elsevier B.V. All rights reserved.)
- Published
- 2018
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26. Enterovirus detection in patients with acute gastroenteritis in Switzerland.
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Gosert R, Heininger U, and Hirsch HH
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- Adolescent, Adult, Aged, Aged, 80 and over, Animals, Bacteria classification, Bacteria isolation & purification, Child, Child, Preschool, Enterovirus genetics, Female, Humans, Infant, Infant, Newborn, Male, Middle Aged, Molecular Diagnostic Techniques, Parasites classification, Parasites isolation & purification, Prevalence, Switzerland epidemiology, Young Adult, Enterovirus classification, Enterovirus isolation & purification, Enterovirus Infections epidemiology, Enterovirus Infections virology, Gastroenteritis epidemiology, Gastroenteritis etiology
- Abstract
Acute gastroenteritis (GE) has a major impact on morbidity and mortality worldwide, yet comprehensive data regarding infectious agents including enteroviruses are scarce. We hypothesized that enteroviruses constitute a significant cause of acute GE. We analyzed 677 stool samples from 504 patients, which had been submitted for suspected infectious GE. 0.2 mL of stool suspension was extracted using the Abbott m2000
sp robot and analysed by multiplex nucleic acid testing (NAT) using the Luminex xTAG gastrointestinal pathogen panel (GPP) as well as by specific NATs detecting enteroviruses and polioviruses. Median age of the patients was 6.6 years (IQR 1.1-50.6; pediatric <18 years). 292 of 677 (43%) samples were positive for at least one pathogen. Enterovirus was detected in 5.3% (36/677) as sole pathogen (67%), and more frequently in children (P = 0.0054). Only rotavirus (18.6%) and norovirus (12.1%) were more frequent. Clostridium difficile and Campylobacter jejuni were detected in 5.5% and 2.2% of stools, respectively. Adenovirus, E. coli O157, Salmonella, Shiga toxin-producing E. coli (STEC), Shigella, Giardia lamblia, Cryptosporidium, and Entamoeba histolytica were rare (<1% of samples). Vibrio cholerae, Yersinia enterocolitica, enterotoxigenic E. coli (ETEC) and poliovirus were not detected. Thus, non-polio enteroviruses are the third most frequent pathogen in acute GE suggesting that enteroviruses may play an important role in GE even in developed, industrial health care settings., (© 2017 Wiley Periodicals, Inc.)- Published
- 2018
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27. Novel Human Polyomavirus Noncoding Control Regions Differ in Bidirectional Gene Expression according to Host Cell, Large T-Antigen Expression, and Clinically Occurring Rearrangements.
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Ajuh ET, Wu Z, Kraus E, Weissbach FH, Bethge T, Gosert R, Fischer N, and Hirsch HH
- Subjects
- HEK293 Cells, Humans, MicroRNAs biosynthesis, MicroRNAs genetics, RNA, Viral biosynthesis, RNA, Viral genetics, Antigens, Viral, Tumor genetics, Antigens, Viral, Tumor metabolism, Gene Expression Regulation, Viral, Gene Rearrangement, Genome, Viral, Models, Genetic, Polyomaviridae genetics, Polyomaviridae metabolism
- Abstract
Human polyomavirus (HPyV) DNA genomes contain three regions denoted the early viral gene region (EVGR), encoding the regulatory T-antigens and one microRNA, the late viral gene region (LVGR), encoding the structural Vp capsid proteins, and the noncoding control region (NCCR). The NCCR harbors the origin of viral genome replication and bidirectional promoter/enhancer functions governing EVGR and LVGR expression on opposite DNA strands. Despite principal similarities, HPyV NCCRs differ in length, sequence, and architecture. To functionally compare HPyV NCCRs, sequences from human isolates were inserted into a bidirectional reporter vector using dsRed2 for EVGR expression and green fluorescent protein (GFP) for LVGR expression. Transfecting HPyV NCCR reporter vectors into human embryonic kidney 293 (HEK293) cells and flow cytometry normalized to archetype BKPyV NCCR revealed a hierarchy of EVGR expression levels with MCPyV, HPyV12, and STLPyV NCCRs conferring stronger levels and HPyV6, HPyV9, and HPyV10 NCCRs weaker levels, while LVGR expression was less variable and showed comparable activity levels. Transfection of HEK293T cells expressing simian virus 40 (SV40) large T antigen (LTag) increased EVGR expression for most HPyV NCCRs, which correlated with the number of LTag-binding sites (Spearman's r , 0.625; P < 0.05) and decreased following SV40 LTag small interfering RNA (siRNA) knockdown. LTag-dependent activation was specifically confirmed for two different MCPyV NCCRs in 293MCT cells expressing the cognate MCPyV LTag. HPyV NCCR expression in different cell lines derived from skin (A375), cervix (HeLaNT), lung (A549), brain (Hs683), and colon (SW480) demonstrated that host cell properties significantly modulate the baseline HPyV NCCR activity, which partly synergized with SV40 LTag expression. Clinically occurring NCCR sequence rearrangements of HPyV7 PITT-1 and -2 and HPyV9 UF1 were found to increase EVGR expression compared to the respective HPyV archetype, but this was partly host cell type specific. IMPORTANCE HPyV NCCRs integrate essential viral functions with respect to host cell specificity, persistence, viral replication, and disease. Here, we show that HPyV NCCRs not only differ in sequence length, number, and position of LTag- and common transcription factor-binding sites but also confer differences in bidirectional viral gene expression. Importantly, EVGR reporter expression was significantly modulated by LTag expression and by host cell properties. Clinical sequence variants of HPyV7 and HPyV9 NCCRs containing deletions and insertions were associated with increased EVGR expression, similar to BKPyV and JCPyV rearrangements, emphasizing that HPyV NCCR sequences are major determinants not only of host cell tropism but also of pathogenicity. These results will help to define secondary HPyV cell tropism beyond HPyV surface receptors, to identify key viral and host factors shaping the viral life cycle, and to develop preclinical models of HPyV persistence and replication and suitable antiviral targets., (Copyright © 2018 American Society for Microbiology.)
- Published
- 2018
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28. Donor-derived, metastatic urothelial cancer after kidney transplantation associated with a potentially oncogenic BK polyomavirus.
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Müller DC, Rämö M, Naegele K, Ribi S, Wetterauer C, Perrina V, Quagliata L, Vlajnic T, Ruiz C, Balitzki B, Grobholz R, Gosert R, Ajuh ET, Hirsch HH, Bubendorf L, and Rentsch CA
- Subjects
- Adult, BK Virus immunology, BK Virus pathogenicity, Cell Transformation, Viral, Child, Preschool, Gene Expression Regulation, Neoplastic, Gene Expression Regulation, Viral, Host-Pathogen Interactions, Humans, Immunocompromised Host, Immunosuppressive Agents adverse effects, Male, Neoplasm Metastasis, Polyomavirus Infections diagnosis, Polyomavirus Infections immunology, Treatment Outcome, Tumor Virus Infections diagnosis, Tumor Virus Infections immunology, Urinary Bladder Neoplasms immunology, Urinary Bladder Neoplasms pathology, Urothelium immunology, Urothelium pathology, BK Virus genetics, Biomarkers, Tumor genetics, Kidney Transplantation adverse effects, Polyomavirus Infections virology, Tissue Donors, Tumor Virus Infections virology, Urinary Bladder Neoplasms genetics, Urinary Bladder Neoplasms virology, Urothelium virology
- Abstract
BK polyomavirus has been linked to urothelial carcinoma in immunosuppressed patients. Here, we performed comprehensive genomic analysis of a BK polyomavirus-associated, metachronous, multifocal and metastatic micropapillary urothelial cancer in a kidney transplant recipient. Dissecting cancer heterogeneity by sorting technologies prior to array-comparative genomic hybridization followed by short tandem repeat analysis revealed that the metastatic urothelial cancer was of donor origin (4-year-old male). The top 50 cancer-associated genes showed no key driver mutations as assessed by next-generation sequencing. Whole genome sequencing and BK polyomavirus-specific amplification provided evidence for episomal and subgenomic chromosomally integrated BK polyomavirus genomes, which carried the same unique 17-bp deletion signature in the viral non-coding control region (NCCR). Whereas no role in oncogenesis could be attributed to the host gene integration in chromosome 1, the 17-bp deletion in the NCCR increased early viral gene expression, but decreased viral replication capacity. Consequently, urothelial cells were exposed to high levels of the transforming BK polyomavirus early proteins large tumour antigen and small tumour antigen from episomal and integrated gene expression. Surgery combined with discontinuation of immunosuppression resulted in complete remission, but sacrificed the renal transplant. Thus, this report links, for the first time, BK polyomavirus NCCR rearrangements with oncogenic transformation in urothelial cancer in immunosuppressed patients. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)
- Published
- 2018
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29. Human BK Polyomavirus Plasmid pBKV (34-2) (Dunlop) Contains Mutations Not Found in the Originally Published Sequences.
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Henriksen S, Mittelholzer C, Gosert R, Hirsch HH, and Rinaldo CH
- Abstract
The plasmid pBKV (34-2) (ATCC 45025) contains the entire BK polyomavirus Dunlop genome. Sequencing revealed 12 point mutations compared to the GenBank sequence, but only 4 point mutations compared to the published sequence. The origin of these differences is unknown, but may impact virological as well as diagnostic research and development., (Copyright © 2015 Henriksen et al.)
- Published
- 2015
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30. Sp1 sites in the noncoding control region of BK polyomavirus are key regulators of bidirectional viral early and late gene expression.
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Bethge T, Hachemi HA, Manzetti J, Gosert R, Schaffner W, and Hirsch HH
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- BK Virus metabolism, DNA, Viral metabolism, Humans, Protein Binding, Viral Proteins metabolism, BK Virus genetics, Gene Expression Regulation, Viral, Polyomavirus Infections virology, Sp1 Transcription Factor metabolism, Transcription, Genetic, Viral Proteins genetics
- Abstract
Unlabelled: In kidney transplant patients with BK polyomavirus (BKPyV) nephropathy, viral variants arise bearing rearranged noncoding control regions (rr-NCCRs) that increase viral early gene expression, replicative fitness, and cytopathology. rr-NCCRs result from various deletions and duplications of archetype NCCR (ww-NCCR) sequences, which alter transcription factor binding sites (TFBS). However, the role of specific TFBS is unclear. We inactivated 28 TFBS in the archetype NCCR by selective point mutations and examined viral gene expression in bidirectional reporter constructs. Compared to the archetype, group 1 mutations increased viral early gene expression similar to rr-NCCR and resulted from inactivating one Sp1 or one Ets1 TFBS near the late transcription start site (TSS). Group 2 mutations conferred intermediate early gene activation and affected NF1, YY1, and p53 sites between early and late TSS. Group 3 mutations decreased early and late gene expression and included two other Sp1 sites near the early TSS. Recombinant viruses bearing group 1 NCCRs showed increased replication in human renal epithelial cells similar to clinical rr-NCCR variants. Group 2 and 3 viruses showed intermediate or no replication, respectively. A literature search revealed unnoticed group 1 mutations in BKPyV nephropathy, hemorrhagic cystitis, and disseminated disease., Importance: The NCCRs of polyomaviruses mediate silent persistence of the viral genome as well as the appropriately timed (re)activation of the viral life cycle. This study indicates that the basal BKPyV NCCR is critically controlled by a hierarchy of single TFBS in the archetype NCCR that direct, modulate, and execute the bidirectional early and late viral gene expression. The results provide new insights into how BKPyV NCCR functions as a viral sensor of host cell signals and shed new light on how transcription factors like Sp1 control bidirectional viral gene expression and contribute to replication and pathology., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)
- Published
- 2015
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31. CMX001 (1-O-hexadecyloxypropyl-cidofovir) inhibits polyomavirus JC replication in human brain progenitor-derived astrocytes.
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Gosert R, Rinaldo CH, Wernli M, Major EO, and Hirsch HH
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- Animals, COS Cells, Cells, Cultured, Chlorocebus aethiops, Cytosine pharmacology, Fluorescent Antibody Technique, Humans, Polymerase Chain Reaction, Astrocytes cytology, Astrocytes virology, Brain cytology, Cytosine analogs & derivatives, JC Virus drug effects, Organophosphonates pharmacology, Stem Cells cytology, Virus Replication drug effects
- Abstract
Polyomavirus JC (JCV) replication causes progressive multifocal leukoencephalopathy (PML), a frequently fatal brain disease in immunodeficient patients, yet antiviral drugs are lacking. We characterized the lipid conjugate 1-O-hexadecyloxypropyl-cidofovir (CMX001) regarding JCV (Mad-4) replication in human brain progenitor-derived astrocytes (PDA) and the simian virus 40 (SV40) large-T-antigen-expressing COS-7 cells up to 7 days postinfection (dpi). We examined JCV loads by PCR, the infection rate by immunofluorescence, and host cell toxicity by WST-1 and BrdU incorporation assays. Supernatants from CMX001-treated PDA demonstrated a drug concentration-dependent decrease in JCV loads and infectivity. CMX001 had only a modest effect on host cell metabolism but reduced overall BrdU incorporation. In PDA at 7 dpi, the CMX001 50% effective concentration (EC50) was 5.55 nM, the 50% cytotoxic concentration (CC50) was 184.6 nM, and the 50% selectivity index (SI50) was 33.3. The EC90 was 19.7 nM, the CC90 was 5,054 nM, and the SI90 was 256.1. In COS-7 cells, JCV replication was faster and the EC50 and EC90 were 18- and 37-fold higher than those in PDA, i.e., 0.1 μM and 0.74 μM (CC50, 0.67 μM; SI50, 6.7; CC90, 12.2 μM; SI90, 16.5) at 5 dpi. We conclude that CMX001 inhibits JCV replication at concentrations in vitro that can be attained by oral administration without significant side effects in clinical studies.
- Published
- 2011
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32. 1-O-hexadecyloxypropyl cidofovir (CMX001) effectively inhibits polyomavirus BK replication in primary human renal tubular epithelial cells.
- Author
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Rinaldo CH, Gosert R, Bernhoff E, Finstad S, and Hirsch HH
- Subjects
- BK Virus genetics, BK Virus metabolism, Blotting, Western, Cell Proliferation drug effects, Cell Survival drug effects, Cells, Cultured, Cytosine pharmacology, Humans, Microscopy, Fluorescence, Reverse Transcriptase Polymerase Chain Reaction, Antiviral Agents pharmacology, BK Virus drug effects, Cytosine analogs & derivatives, Organophosphonates pharmacology, Virus Replication drug effects
- Abstract
Antiviral drugs for treating polyomavirus BK (BKV) replication in polyomavirus-associated nephropathy or hemorrhagic cystitis are of considerable clinical interest. Unlike cidofovir, the lipid conjugate 1-O-hexadecyloxypropyl cidofovir (CMX001) is orally available and has not caused detectable nephrotoxicity in rodent models or human studies to date. Primary human renal proximal tubular epithelial cells were infected with BKV-Dunlop, and CMX001 was added 2 h postinfection (hpi). The intracellular and extracellular BKV DNA load was determined by quantitative PCR. Viral gene expression was examined by quantitative reverse transcription-PCR, Western blotting, and immunofluorescence microscopy. We also examined host cell viability, proliferation, metabolic activity, and DNA replication. The titration of CMX001 identified 0.31 μM as the 90% effective concentration (EC(90)) for reducing the extracellular BKV load at 72 hpi. BKV large T antigen mRNA and protein expression was unaffected at 24 hpi, but the intracellular BKV genome was reduced by 90% at 48 hpi. Late gene expression was reduced by 70 and 90% at 48 and 72 hpi, respectively. Comparisons of CMX001 and cidofovir EC(90)s from 24 to 96 hpi demonstrated that CMX001 had a more rapid and enduring effect on BKV DNA and infectious progeny at 96 hpi than cidofovir. CMX001 at 0.31 μM had little effect on overall cell metabolism but reduced bromodeoxyuridine incorporation and host cell proliferation by 20 to 30%, while BKV infection increased cell proliferation in both rapidly dividing and near-confluent cultures. We conclude that CMX001 inhibits BKV replication with a longer-lasting effect than cidofovir at 400× lower levels, with fewer side effects on relevant host cells in vitro.
- Published
- 2010
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33. Rearranged JC virus noncoding control regions found in progressive multifocal leukoencephalopathy patient samples increase virus early gene expression and replication rate.
- Author
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Gosert R, Kardas P, Major EO, and Hirsch HH
- Subjects
- Adult, Animals, Base Sequence, COS Cells, Chlorocebus aethiops, DNA Primers genetics, DNA, Viral cerebrospinal fluid, DNA, Viral genetics, DNA, Viral urine, Female, Gene Expression, Gene Rearrangement, Genes, Reporter, Genes, Viral, Humans, JC Virus isolation & purification, JC Virus pathogenicity, Male, Middle Aged, Transfection, Virus Replication, JC Virus genetics, JC Virus physiology, Leukoencephalopathy, Progressive Multifocal virology
- Abstract
Polyomavirus JC (JCV) infects ∼ 60% of the general population, followed by asymptomatic urinary shedding in ∼ 20%. In patients with pronounced immunodeficiency, including HIV/AIDS, JCV can cause progressive multifocal leukoencephalopathy (PML), a devastating brain disease of high mortality. While JCV in the urine of healthy people has a linear noncoding control region called the archetype NCCR (at-NCCR), JCV in brain and cerebrospinal fluid (CSF) of PML patients bear rearranged NCCRs (rr-NCCRs). Although JCV NCCR rearrangements are deemed pathognomonic for PML, their role as a viral determinant is unclear. We sequenced JCV NCCRs found in CSF of eight HIV/AIDS patients newly diagnosed with PML and analyzed their effect on early and late gene expression using a bidirectional reporter vector recapitulating the circular polyomavirus early and late gene organization. The rr-NCCR sequences were highly diverse, but all increased viral early reporter gene expression in progenitor-derived astrocytes, glia-derived cells, and human kidney compared to the expression levels with the at-NCCR. The expression of simian virus 40 (SV40) large T antigen or HIV Tat expression in trans was associated with a strong increase of at-NCCR-controlled early gene expression, while rr-NCCRs were less responsive. The insertion of rr-NCCRs into the JCV genome backbone revealed higher viral replication rates for rr-NCCR compared to those of the at-NCCR JCV in human progenitor-derived astrocytes or glia cells, which was abrogated in SV40 large T-expressing COS-7 cells. We conclude that naturally occurring JCV rr-NCCR variants from PML patients confer increased early gene expression and higher replication rates compared to those of at-NCCR JCV and thereby increase cytopathology.
- Published
- 2010
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34. The polyomavirus BK agnoprotein co-localizes with lipid droplets.
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Unterstab G, Gosert R, Leuenberger D, Lorentz P, Rinaldo CH, and Hirsch HH
- Subjects
- Amino Acid Sequence, Animals, Cell Nucleus chemistry, Cells, Cultured, Chlorocebus aethiops, Cytoplasm chemistry, Humans, Microscopy, Confocal, Microscopy, Fluorescence, Molecular Sequence Data, Mutation, Phosphorylation, Protein Structure, Secondary, Vero Cells, BK Virus chemistry, Lipids chemistry, Viral Regulatory and Accessory Proteins chemistry
- Abstract
Agnoprotein encoded by human polyomavirus BK (BKV) is a late cytoplasmic protein of 66 amino acids (aa) of unknown function. Immunofluorescence microscopy revealed a fine granular and a vesicular distribution in donut-like structures. Using BKV(Dunlop)-infected or agnoprotein-transfected cells, we investigated agnoprotein co-localization with subcellular structures. We found that agnoprotein co-localizes with lipid droplets (LD) in primary human renal tubular epithelial cells as well as in other cells supporting BKV replication in vitro (UTA, Vero cells). Using agnoprotein-enhanced green fluorescent protein (EGFP) fusion constructs, we demonstrate that agnoprotein aa 20-42 are required for targeting LD, whereas aa 1-20 or aa 42-66 were not. Agnoprotein aa 22-40 are predicted to form an amphipathic helix, and mutations A25D and F39E, disrupting its hydrophobic domain, prevented LD targeting. However, changing the phosphorylation site serine-11 to alanine or aspartic acid did not alter LD co-localization. Our findings provide new clues to unravel agnoprotein function., (Copyright 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
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35. Clinical polyomavirus BK variants with agnogene deletion are non-functional but rescued by trans-complementation.
- Author
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Myhre MR, Olsen GH, Gosert R, Hirsch HH, and Rinaldo CH
- Subjects
- Animals, Burkitt Lymphoma, DNA, Viral, Gene Deletion, Gene Expression Regulation, Viral physiology, Humans, Promoter Regions, Genetic genetics, BK Virus genetics, Viral Regulatory and Accessory Proteins genetics, Viral Regulatory and Accessory Proteins metabolism, Virus Replication genetics
- Abstract
High-level replication of polyomavirus BK (BKV) in kidney transplant recipients is associated with the emergence of BKV variants with rearranged (rr) non-coding control region (NCCR) increasing viral early gene expression and cytopathology. Cloning and sequencing revealed the presence of a BKV quasispecies which included non-functional variants when assayed in a recombinant virus assay. Here we report that the rr-NCCR of BKV variants RH-3 and RH-12, both bearing a NCCR deletion including the 5' end of the agnoprotein coding sequence, mediated early and late viral reporter gene expression in kidney cells. However, in a recombinant virus they failed to produce infectious progeny despite large T-antigen and VP1 expression and the formation of nuclear virus-like particles. Infectious progeny was generated when the agnogene was reconstructed in cis or agnoprotein provided in trans from a co-existing BKV rr-NCCR variant. We conclude that complementation can rescue non-functional BKV variants in vitro and possibly in vivo., (Copyright 2009 Elsevier Inc. All rights reserved.)
- Published
- 2010
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36. Antibody responses to recombinant polyomavirus BK large T and VP1 proteins in young kidney transplant patients.
- Author
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Bodaghi S, Comoli P, Bösch R, Azzi A, Gosert R, Leuenberger D, Ginevri F, and Hirsch HH
- Subjects
- Adolescent, Adult, Animals, Baculoviridae genetics, Blood virology, Child, Child, Preschool, Genetic Vectors, Humans, Kidney Transplantation, Polyomavirus Infections virology, Recombinant Proteins genetics, Urine virology, Young Adult, Antibodies, Viral blood, Antigens, Polyomavirus Transforming genetics, BK Virus immunology, Capsid Proteins genetics, Enzyme-Linked Immunosorbent Assay methods, Hemagglutination Inhibition Tests methods, Polyomavirus Infections immunology
- Abstract
BK virus (BKV)-specific immunity is critical for polyomavirus-associated nephropathy, but antibody responses are incompletely defined. We compared the hemagglutination inhibition assay (HIA) with immunoglobulin G enzyme immunoassays (EIA) to BKV proteins expressed in baculovirus-infected insect cells. N-terminal, internal, and C-terminal domains of the BKV large T antigen (BKLT) were fused to glutathione S-transferase (GST), yielding GST-BKLTD1, GST-BKLTD2, and GST-BKLTD3, respectively. The BKV capsid VP1 was expressed as a GST fusion (BKVP1) or as a native VP1 assembled into viruslike particles (BKVLP). We tested 422 sera from 28 healthy donors (HD), 99 dialysis patients (DP; median age, 15 years; range, 3 to 32 years), and 46 age-matched kidney transplant patients (KTP; median age, 15 years; range, 2 to 33 years). In HD, HIA and BKVLP EIA both yielded a 91.7% seroreactivity, whereas all other EIA responses were lower (BKVP1, 83.3%; BKLTD1, 25%; BKLTD2, 29%; BKLTD3, 40%). HIA titers significantly correlated with EIA levels for BKVLP, BKVP1, and BKLTD1 but not for BKLTD2 or BKLTD3, which were barely above the cutoff. In DP, the seroreactivities of HIA, BKVLP, and BKLTD1 were lower than that in HD (63.6%, 86.9%, and 10.1%, respectively) and they had lower titers (P < 0.001). In KTP, seropositivities for BKVLP, BKVP1, and BKLTD1 were 78%, 50%, and 17%, respectively, but anti-BKVLP levels increased significantly in KTP with viruria and viremia, whereas anti-BKLTD1 levels increased after clearing sustained BKV viremia. In conclusion, anti-BKVLP is equivalent to HIA in HD but is more sensitive to determine the BKV serostatus in DP and KTP. In KTP, anti-BKVLP responds to recent BKV viruria and viremia, whereas anti-BKLTD1 may indicate emerging BKV-specific immune control.
- Published
- 2009
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37. Prevalence of polyomavirus BK and JC infection and replication in 400 healthy blood donors.
- Author
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Egli A, Infanti L, Dumoulin A, Buser A, Samaridis J, Stebler C, Gosert R, and Hirsch HH
- Subjects
- Adult, Capsid Proteins genetics, Capsid Proteins immunology, Female, Humans, Male, Middle Aged, Polymerase Chain Reaction, Polyomavirus Infections diagnosis, Prevalence, Seroepidemiologic Studies, Sex Characteristics, Switzerland, Urine virology, Young Adult, BK Virus genetics, BK Virus physiology, Blood Donors, JC Virus genetics, JC Virus physiology, Polyomavirus Infections epidemiology, Virus Replication
- Abstract
Background: The replication of BK virus (BKV) and JC virus (JCV) is linked to polyomavirus-associated nephropathy, hemorrhagic cystitis, and multifocal leukoencephalopathy in immunodeficient patients, but the behavior of these viruses in immunocompetent individuals has hardly been characterized., Methods: We used EIA to study samples obtained from 400 healthy blood donors aged 20-59 years for BKV- and JCV-specific antibodies against virus-like particles. We also studied BKV and JCV loads in plasma and urine among these individuals by use of real-time polymerase chain reaction., Results: IgG seroprevalence was 82% (328 of 400 donors) for BKV and 58% (231 of400) for JCV. As age increased (age groups were divided by decade), the seroprevalence of BKV decreased from 87% (87 of 100) in the youngest group (aged 20-29 years) to 71% (71 of 100) in the oldest group (aged 50-59 years) (P = .006), whereas the seroprevalence of JCV increased from 50% (50 of 100) in the youngest group to 68% (68 of 100) in the oldest group (P = .06). Asymptomatic urinary shedding of BKV and JCV was observed in 28 (7%) and 75 (19%) of 400 subjects, respectively, with median viral loads of 3.51 and 4.64 log copies/mL, respectively (P < .001). Unlike urinary BKV loads, urinary JCV loads were positively correlated with IgG levels. The shedding of JCV was more commonly observed among individuals who were seropositive only for JCV, compared with individuals who were seropositive for both BKV and JCV, suggesting limited cross-protection from BKV immunity. Noncoding control regions were of archetype architecture in all cases, except for 1 rearranged JCV variant. Neither BKV nor JCV DNA was detected in plasma., Conclusions: Our study provides important data about polyomavirus infection and replication in healthy, immunocompetent individuals. These data indicate significant differences between BKV and JCV with respect to virus-host interaction and epidemiology.
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- 2009
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38. Investigation of the hepatitis C virus replication complex.
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Brass V, Gosert R, and Moradpour D
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- Animals, Bromodeoxyuridine metabolism, Cell Membrane metabolism, Fluorescent Antibody Technique, In Situ Hybridization, Fluorescence, Protein Biosynthesis, Staining and Labeling, Transcription, Genetic, Viral Proteins biosynthesis, Hepacivirus metabolism, RNA, Viral biosynthesis, Viral Proteins chemistry, Viral Proteins metabolism, Virus Replication
- Abstract
Formation of a membrane-associated replication complex, composed of viral proteins, replicating RNA, altered cellular membranes, and other host factors, is a hallmark of all positive-strand RNA viruses. In the case of HCV, RNA replication takes place in a likely endoplasmic reticulum-derived membrane alteration referred to as the "membranous web." In vitro transcription-translation, membrane extraction and flotation analyses, immunofluorescence microscopy, fluorescent in situ hybridization, and RNA metabolic labeling followed by confocal laser scanning microscopy have yielded insights into the structure and function of the HCV replication complex. We describe these techniques and highlight selected results.
- Published
- 2009
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39. Polyomavirus BK with rearranged noncoding control region emerge in vivo in renal transplant patients and increase viral replication and cytopathology.
- Author
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Gosert R, Rinaldo CH, Funk GA, Egli A, Ramos E, Drachenberg CB, and Hirsch HH
- Subjects
- Adolescent, Adult, Aged, Animals, Cell Line, Cytopathogenic Effect, Viral, DNA, Viral blood, DNA, Viral urine, Disease Progression, Female, Gene Expression Regulation, Genes, Reporter, Humans, Male, Middle Aged, Polyomavirus Infections pathology, Polyomavirus Infections virology, Tumor Virus Infections pathology, Tumor Virus Infections virology, Viral Fusion Proteins metabolism, Viral Load, Viremia, BK Virus genetics, BK Virus physiology, DNA, Viral genetics, Kidney Transplantation, Regulatory Sequences, Nucleic Acid genetics, Virus Replication
- Abstract
Immunosuppression is required for BK viremia and polyomavirus BK-associated nephropathy (PVAN) in kidney transplants (KTs), but the role of viral determinants is unclear. We examined BKV noncoding control regions (NCCR), which coordinate viral gene expression and replication. In 286 day-matched plasma and urine samples from 129 KT patients with BKV viremia, including 70 with PVAN, the majority of viruses contained archetypal (ww-) NCCRs. However, rearranged (rr-) NCCRs were more frequent in plasma than in urine samples (22 vs. 4%; P < 0.001), and were associated with 20-fold higher plasma BKV loads (2.0 x 10(4)/ml vs. 4.4 x 10(5)/ml; P < 0.001). Emergence of rr-NCCR in plasma correlated with duration and peak BKV load (R(2) = 0.64; P < 0.001). This was confirmed in a prospective cohort of 733 plasma samples from 227 patients. For 39 PVAN patients with available biopsies, rr-NCCRs were associated with more extensive viral replication and inflammation. Cloning of 10 rr-NCCRs revealed diverse duplications or deletions in different NCCR subregions, but all were sufficient to increase early gene expression, replication capacity, and cytopathology of recombinant BKV in vitro. Thus, rr-NCCR BKV emergence in plasma is linked to increased replication capacity and disease in KTs.
- Published
- 2008
- Full Text
- View/download PDF
40. Polyomavirus BK versus JC replication and nephropathy in renal transplant recipients: a prospective evaluation.
- Author
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Drachenberg CB, Hirsch HH, Papadimitriou JC, Gosert R, Wali RK, Munivenkatappa R, Nogueira J, Cangro CB, Haririan A, Mendley S, and Ramos E
- Subjects
- Adult, Aged, Aged, 80 and over, BK Virus genetics, Base Sequence, Biopsy, Cohort Studies, DNA, Viral genetics, Female, Humans, Incidence, JC Virus genetics, Kidney pathology, Kidney virology, Male, Middle Aged, Molecular Sequence Data, Polyomavirus Infections pathology, Prospective Studies, Tumor Virus Infections pathology, BK Virus physiology, JC Virus physiology, Kidney Diseases pathology, Kidney Diseases virology, Kidney Transplantation pathology, Virus Replication physiology
- Abstract
Background: JC virus (JCV) viruria is more common than BK virus (BKV) viruria in healthy individuals but in kidney transplants (KT), polyomavirus nephropathy (PVAN) is primarily caused by BKV. Few cases of PVAN have been attributed to JCV. Systematic studies on JCV replication in KT are lacking., Methods: Out of a cohort of KT patients screened with urine cytology, patients shedding decoy cells were studied (n=103). Molecular studies demonstrated BKV, JCV, or BKV+JCV shedding in 58 (56.3%), 28 (27.2%), and 17 (16.5%), respectively. Biopsy was performed when decoy cells persisted 2 months or serum creatinine increased >20%., Results: BKV viruria was strongly associated with BKV viremia (93%), PVAN (48%, P=0.01) and graft loss (P=0.03). Higher BKV viremia correlated with graft dysfunction (P=0.01), more advanced histological pattern of PVAN (P<0.0001), and more infected cells in biopsy (P=0.0001). BKV viremia of > or =10,000 copies/mL was significantly associated with histologically confirmed PVAN (P=0.0001). Reduction of immunosuppression lead to disappearance of decoy cells in patients shedding BK (>93%). JCV viruria, was more often asymptomatic (P=0.002) and affected older patients (P=0.02). JCV PVAN was less common (21.4%) and was characterized by sparse cytopathic changes but significant inflammation and fibrosis. JCV viremia was rare (14.2%), transient, and low (mean 2.0E+03/mL). After reduction of immunosuppression decoy cells persisted in >50% of patients with JCV (P=0.0001), but no graft loss occurred. During the period of the current study, the incidence of BKV-PVAN was 5.5% and the incidence of JCV-PVAN was 0.9%., Conclusions: The data point to significant differences of BKV and JCV biology regarding replication and disease in KT patients, with important implications for screening and management.
- Published
- 2007
- Full Text
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41. Human polyomavirus type 1 (BK virus) agnoprotein is abundantly expressed but immunologically ignored.
- Author
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Leuenberger D, Andresen PA, Gosert R, Binggeli S, Ström EH, Bodaghi S, Rinaldo CH, and Hirsch HH
- Subjects
- Antigens, Viral, Tumor immunology, Biopsy, Epithelial Cells metabolism, Humans, Immunity, Cellular, Interferon-gamma biosynthesis, Kidney Diseases virology, Kidney Tubules, Proximal cytology, Leukocytes, Mononuclear immunology, Polyomavirus Infections immunology, Polyomavirus Infections virology, Recombinant Proteins genetics, Recombinant Proteins immunology, Recombinant Proteins metabolism, Tumor Virus Infections immunology, Tumor Virus Infections virology, Viral Proteins genetics, Viral Proteins isolation & purification, Viral Regulatory and Accessory Proteins, Virus Replication, Antibodies, Viral blood, BK Virus immunology, Kidney Diseases immunology, Kidney Transplantation immunology, Kidney Tubules, Proximal metabolism, Viral Proteins immunology, Viral Proteins metabolism
- Abstract
Impaired BK virus (BKV)-specific immunity is a key risk factor of polyomavirus-associated nephropathy. We hypothesized that BKV agnoprotein might constitute an important immune target, as it is highly expressed after infection in vitro. We demonstrate abundant expression of BKV agnoprotein in vivo by immunostaining of kidney transplant (KT) biopsy specimens. Antibody responses to the recombinant affinity-purified BKV agnoprotein, large tumor (LT), and VP1 antigens in 146 sera from 38 KT patients and in 19 sera from 16 healthy donors (HD) were compared by enzyme immunoassay. In HD, low titers of anti-agnoprotein immunoglobulin G (IgG) were found in 15% of sera, compared to 41% for anti-LT antigen and 63% for anti-VP1. No anti-BKV IgM was detectable. In KT patients, anti-agnoprotein IgG and IgM were found in 8% and 3.6% of sera, compared to 63% and 18% for anti-LT IgG and IgM and 80% and 41% for anti-VP1 IgG and IgM, respectively. Anti-LT antigen and anti-VP1, but not anti-agnoprotein, activities increased during and after BKV viremia in KT patients. To investigate specific cellular immune responses, we compared levels of gamma interferon production in peripheral blood mononuclear cells (PBMC) of 10 HD and 30 KT patients by enzyme-linked immunospot assay. In HD, the median numbers of gamma interferon spot-forming units per million PBMC for the agnoprotein, LT antigen, and VP1 peptides were 1, 23, and 25, respectively, whereas the responses in KT patients were 2, 24, and 99, respectively. We conclude that BKV agnoprotein, though abundantly expressed in vivo, is poorly recognized immunologically.
- Published
- 2007
- Full Text
- View/download PDF
42. Viral dynamics in transplant patients: implications for disease.
- Author
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Funk GA, Gosert R, and Hirsch HH
- Subjects
- Humans, Virus Diseases virology, Transplantation, Viral Load, Virus Diseases diagnosis, Virus Diseases drug therapy, Viruses growth & development
- Abstract
Viral infections cause substantial morbidity and mortality in transplant patients. Quantifying viral loads is widely appreciated as a direct means to diagnose and monitor the course of viral infections. Recent studies indicate that the kinetics of viral load changes rather than single viral load measurements better correlate with organ involvement. In this Review, we will summarise the current knowledge regarding the kinetics of viruses relevant to transplantation including cytomegalovirus, Epstein-Barr virus, the herpes viruses 6 and 7, hepatitis C virus, GB virus C, adenovirus, and the emerging human polyomavirus type BK. We discuss the implications of viral kinetics for organ pathology as well as for the evaluation of antiviral interventions in transplant patients.
- Published
- 2007
- Full Text
- View/download PDF
43. Characterization of highly frequent epitope-specific CD45RA+/CCR7+/- T lymphocyte responses against p53-binding domains of the human polyomavirus BK large tumor antigen in HLA-A*0201+ BKV-seropositive donors.
- Author
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Provenzano M, Bracci L, Wyler S, Hudolin T, Sais G, Gosert R, Zajac P, Palu' G, Heberer M, Hirsch HH, and Spagnoli GC
- Abstract
Human polyomavirus BK (BKV) has been implicated in oncogenic transformation. Its ability to replicate is determined by the binding of its large tumor antigen (LTag) to products of tumor-suppressor genes regulating cell cycle, as specifically p53. We investigated CD8+ T immune responses to BKV LTag portions involved in p53 binding in HLA-A*0201+ BKV LTag experienced individuals. Peptides selected from either p53-binding region (LTag351-450 and LTag533-626) by current algorithms and capacity to bind HLA-A*0201 molecule were used to stimulate CD8+ T responses, as assessed by IFN-gamma gene expression ex vivo and detected by cytotoxicity assays following in vitro culture. We observed epitope-specific immune responses in all HLA-A*0201+ BKV LTag experienced individuals tested. At least one epitope, LTag579-587; LLLIWFRPV, was naturally processed in non professional antigen presenting cells and induced cytotoxic responses with CTL precursor frequencies in the order of 1/20'000. Antigen specific CD8+ T cells were only detectable in the CD45RA+ subset, in both CCR7+ and CCR7- subpopulations. These data indicate that widespread cellular immune responses against epitopes within BKV LTag-p53 binding regions exist and question their roles in immunosurveillance against tumors possibly associated with BKV infection.
- Published
- 2006
- Full Text
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44. Characterization of nonstructural protein membrane anchor deletion mutants expressed in the context of the hepatitis C virus polyprotein.
- Author
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Gosert R, Jendrsczok W, Berke JM, Brass V, Blum HE, and Moradpour D
- Subjects
- Cell Line, Tumor, Cell Membrane metabolism, Hepacivirus genetics, Hepacivirus metabolism, Humans, Membrane Microdomains, Mutation, Subcellular Fractions metabolism, Viral Nonstructural Proteins genetics, Virus Replication, Gene Deletion, Hepacivirus physiology, Polyproteins metabolism, Viral Nonstructural Proteins metabolism
- Abstract
Protein-protein interactions involved in formation of the membrane-associated hepatitis C virus (HCV) replication complex are poorly understood. Here, we investigated nonstructural proteins with deletions in their membrane anchor domains when expressed in the context of the entire HCV polyprotein. Interactions among cytosolic domains of HCV nonstructural proteins were found not to be sufficiently strong to rescue such mutants to the membrane. Thus, the membrane anchor domains of nonstructural proteins are essential for incorporation of these proteins into the HCV replication complex while interactions among the cytosolic domains appear to be relatively weak. This feature may provide the nonstructural proteins with a certain flexibility to perform their multiple functions during HCV replication.
- Published
- 2005
- Full Text
- View/download PDF
45. Replication and in vivo repair of the hepatitis A virus genome lacking the poly(A) tail.
- Author
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Kusov YY, Gosert R, and Gauss-Müller V
- Subjects
- Cell Cycle physiology, Cells, Cultured, Hepatitis A virus genetics, Humans, Picornaviridae, Genome, Viral, Hepatitis A virus physiology, RNA, Messenger genetics, RNA, Messenger metabolism, RNA, Viral genetics, RNA, Viral metabolism, Virus Replication
- Abstract
The precise role of the poly(A) tail at the 3' end of the picornavirus RNA genome and the cellular factors that control its homeostasis are unknown. To assess the importance of the poly(A) tail for virus replication, the genome of the slowly replicating hepatitis A virus (HAV) with and without a poly(A) tail was studied after transfection into cells maintained under various conditions. A tailless HAV genome had a shorter half-life than a poly(A)-containing genome and was unable to replicate in quiescent cells. In dividing cells, the tailless RNA gave rise to infectious virus with a restored poly(A) tail of up to 60 residues. Cells arrested at the G(0) and the G(2)/M phase produced lower amounts of infectious HAV than cells in the G(1) phase. These data suggest that the 3' poly(A) tail of HAV can be restored with the help of a cellular and/or viral function that is regulated during the cell cycle.
- Published
- 2005
- Full Text
- View/download PDF
46. Membrane association of the RNA-dependent RNA polymerase is essential for hepatitis C virus RNA replication.
- Author
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Moradpour D, Brass V, Bieck E, Friebe P, Gosert R, Blum HE, Bartenschlager R, Penin F, and Lohmann V
- Subjects
- Amino Acid Sequence, Base Sequence, Viral Nonstructural Proteins chemistry, Virus Replication, Hepacivirus genetics, RNA, Viral biosynthesis, RNA-Dependent RNA Polymerase physiology, Viral Nonstructural Proteins physiology
- Abstract
The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), belongs to a class of integral membrane proteins termed tail-anchored proteins. Its membrane association is mediated by the C-terminal 21 amino acid residues, which are dispensable for RdRp activity in vitro. For this study, we investigated the role of this domain, termed the insertion sequence, in HCV RNA replication in cells. Based on a structural model and the amino acid conservation among different HCV isolates, we designed a panel of insertion sequence mutants and analyzed their membrane association and RNA replication. Subgenomic replicons with a duplication of an essential cis-acting replication element overlapping the sequence that encodes the C-terminal domain of NS5B were used to unequivocally distinguish RNA versus protein effects of these mutations. Our results demonstrate that the membrane association of the RdRp is essential for HCV RNA replication. Interestingly, certain amino acid substitutions within the insertion sequence abolished RNA replication without affecting membrane association, indicating that the C-terminal domain of NS5B has functions beyond serving as a membrane anchor and that it may be involved in critical intramembrane protein-protein interactions. These results have implications for the functional architecture of the HCV replication complex and provide new insights into the expanding spectrum of tail-anchored proteins.
- Published
- 2004
- Full Text
- View/download PDF
47. Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes.
- Author
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Moradpour D, Evans MJ, Gosert R, Yuan Z, Blum HE, Goff SP, Lindenbach BD, and Rice CM
- Subjects
- Amino Acid Sequence, Cell Line, Tumor, DNA Transposable Elements, Green Fluorescent Proteins, Hepacivirus genetics, Hepacivirus metabolism, Humans, Luminescent Proteins metabolism, Microscopy, Fluorescence, Molecular Sequence Data, Mutagenesis, Insertional, RNA, Viral biosynthesis, RNA-Dependent RNA Polymerase, Viral Nonstructural Proteins chemistry, Viral Nonstructural Proteins metabolism, Hepacivirus ultrastructure, Luminescent Proteins genetics, Recombinant Fusion Proteins metabolism, Replicon physiology, Viral Nonstructural Proteins genetics
- Abstract
Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.
- Published
- 2004
- Full Text
- View/download PDF
48. Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons.
- Author
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Gosert R, Egger D, Lohmann V, Bartenschlager R, Blum HE, Bienz K, and Moradpour D
- Subjects
- Cell Membrane metabolism, Cell Membrane ultrastructure, Cell Membrane virology, Hepacivirus metabolism, Hepacivirus ultrastructure, Humans, RNA, Viral genetics, Tumor Cells, Cultured, Viral Nonstructural Proteins metabolism, Hepacivirus genetics, RNA, Viral biosynthesis, RNA, Viral ultrastructure, Replicon, Virus Replication
- Abstract
Formation of a membrane-associated replication complex, composed of viral proteins, replicating RNA, and altered cellular membranes, is a characteristic feature of plus-strand RNA viruses. Here, we demonstrate the presence of a specific membrane alteration, designated the membranous web, that contains hepatitis C virus (HCV) nonstructural proteins, as well as viral plus-strand RNA, in Huh-7 cells harboring autonomously replicating subgenomic HCV RNAs. Metabolic labeling with 5-bromouridine 5'-triphosphate in the presence of actinomycin D revealed that the membranous web is the site of viral RNA synthesis and therefore represents the replication complex of HCV.
- Published
- 2003
- Full Text
- View/download PDF
49. [Molecular virology of hepatitis C].
- Author
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Moradpour D, Brass V, Gosert R, Wölk B, and Blum HE
- Subjects
- Animals, Antiviral Agents therapeutic use, Cells, Cultured, Disease Models, Animal, Enzyme Inhibitors therapeutic use, Forecasting, Genes, Viral, Genetic Therapy, Hepacivirus enzymology, Hepatitis C complications, Hepatitis C drug therapy, Hepatocytes virology, Humans, Mice, Mice, Transgenic, Pan troglodytes, Polyproteins genetics, Polyproteins metabolism, Protease Inhibitors therapeutic use, Protein Biosynthesis, RNA, Viral genetics, Replicon genetics, Transcription, Genetic, Tumor Cells, Cultured, Tupaia, Viral Proteins genetics, Viral Proteins metabolism, Virus Replication, Hepacivirus genetics, Hepacivirus physiology, Hepatitis C therapy, Hepatitis C virology
- Abstract
Hepatitis C virus (HCV) infection is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide. Here, we will briefly review current concepts of the molecular virology of hepatitis C. In vitro and in vivo models of HCV replication will be discussed in this context. Finally, novel antiviral strategies will be outlined that result from an improved understanding of the viral life cycle.
- Published
- 2002
- Full Text
- View/download PDF
50. Hepatitis C: molecular virology and antiviral targets.
- Author
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Moradpour D, Brass V, Gosert R, Wölk B, and Blum HE
- Subjects
- Animals, Genes, Viral, Hepacivirus metabolism, Hepacivirus ultrastructure, Hepatitis C drug therapy, Hepatitis C metabolism, Humans, Models, Molecular, Protein Processing, Post-Translational, Protein Structure, Quaternary, Viral Structural Proteins chemistry, Viral Structural Proteins metabolism, Antiviral Agents therapeutic use, Hepacivirus genetics, Hepatitis C therapy, Hepatitis C virology
- Abstract
Chronic hepatitis C is a leading cause of liver cirrhosis and hepatocellular carcinoma worldwide. Although current treatment options are limited, progress in understanding the molecular virology of hepatitis C has led to the identification of novel antiviral targets. Moreover, in vitro and in vivo model systems have been developed that allow systematic evaluation of new therapeutic strategies. This review details current concepts in molecular virology and emerging therapies for hepatitis C.
- Published
- 2002
- Full Text
- View/download PDF
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