Publications by authors named "Ulrike Protzer"

182 Publications

Immunocompromised patients with therapy-refractory chronic skin diseases show reactivation of latent EBV and CMV infection.

J Invest Dermatol 2021 Sep 1. Epub 2021 Sep 1.

Department of Dermatology and Allergy, Technical University of Munich, Munich, Germany; Division of Dermatology and Venereology, Department of Medicine Solna, and Center for molecular medicine, Karolinska Institutet; Stockholm, Sweden;. Electronic address:

Reactivation of latent Epstein-Barr virus (EBV) and/or Cytomegalovirus (CMV) infection is a dreaded complication in immunocompromised patients receiving hematopoietic stem cell transplantation. Evidence is sparse if subclinical reactivation of viral infection may also be of clinical relevance in dermatological patients. We screened patients (n= 206) suffering from chronic skin diseases for subclinical reactivation of EBV and CMV infection. We found that immunocompromised patients with therapy-refractory chronic skin diseases showed higher rates of subclinical reactivation of CMV and EBV infection (6.7 % vs. 0 % for EBV and 16.7 % vs. 5.6% for CMV) and higher prevalence of virus specific DNA in skin tissue (30.8 % vs. 0% for EBV and 21.4% vs. 0% for CMV) as compared to non-immunocompromised patients with chronic skin diseases. T cells isolated from lesional skin exhibited up to 14-fold increased proliferation with production of Th1 and Th17 cytokines upon stimulation with viral proteins providing evidence for possible aggravation of the underlying skin diseases by viral infection. Improvement of skin lesions in patients with reactivation of CMV infection (n=4) was observed upon anti-viral treatment. Our data suggests that subclinical reactivation of EBV and/or CMV infection is an under-recognized condition in the dermatological patient population with chronic skin diseases.
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http://dx.doi.org/10.1016/j.jid.2021.07.171DOI Listing
September 2021

Targeted T cell receptor gene editing provides predictable T cell product function for immunotherapy.

Cell Rep Med 2021 Aug 17;2(8):100374. Epub 2021 Aug 17.

Institute for Medical Microbiology, Immunology and Hygiene, Technical University of Munich (TUM), Munich, Germany.

Adoptive transfer of T cells expressing a transgenic T cell receptor (TCR) has the potential to revolutionize immunotherapy of infectious diseases and cancer. However, the generation of defined TCR-transgenic T cell medicinal products with predictable function still poses a major challenge and limits broader and more successful application of this "living drug." Here, by studying 51 different TCRs, we show that conventional genetic engineering by viral transduction leads to variable TCR expression and functionality as a result of variable transgene copy numbers and untargeted transgene integration. In contrast, CRISPR/Cas9-mediated TCR replacement enables defined, targeted TCR transgene insertion into the TCR gene locus. Thereby, T cell products display more homogeneous TCR expression similar to physiological T cells. Importantly, increased T cell product homogeneity after targeted TCR gene editing correlates with predictable T cell responses, which represents a crucial aspect for clinical application in adoptive T cell immunotherapy.
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http://dx.doi.org/10.1016/j.xcrm.2021.100374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8385324PMC
August 2021

Rapid and Robust Continuous Purification of High-Titer Hepatitis B Virus for In Vitro and In Vivo Applications.

Viruses 2021 Jul 30;13(8). Epub 2021 Jul 30.

Institute of Virology, Technische Universität München/Helmholtz Zentrum München, 81675 Munich, Germany.

Available treatments for hepatitis B can control the virus but are rarely curative. This led to a global initiative to design new curative therapies for the 257 million patients affected. Discovery and development of these new therapies is contingent upon functional in vitro and in vivo hepatitis B virus (HBV) infection models. However, low titer and impurity of conventional HBV stocks reduce significance of in vitro infections and moreover limit challenge doses in current in vivo models. Therefore, there is a critical need for a robust, simple and reproducible protocol to generate high-purity and high-titer infectious HBV stocks. Here, we outline a three-step protocol for continuous production of high-quality HBV stocks from supernatants of HBV-replicating cell lines. This purification process takes less than 6 h, yields to high-titer stocks (up to 1 × 10 enveloped, DNA-containing HBV particles/mL each week), and is with minimal equipment easily adaptable to most laboratory settings.
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http://dx.doi.org/10.3390/v13081503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8402639PMC
July 2021

Immunogenicity and Antiviral Response of Therapeutic Hepatitis B Vaccination in a Mouse Model of HBeAg-Negative, Persistent HBV Infection.

Vaccines (Basel) 2021 Jul 31;9(8). Epub 2021 Jul 31.

Institute of Virology, Technical University of Munich/Helmholtz Zentrum München, D-81675 Munich, Germany.

During the natural course of chronic hepatitis B virus (HBV) infection, the hepatitis B e antigen (HBeAg) is typically lost, while the direct transmission of HBeAg-negative HBV may result in fulminant hepatitis B. While the induction of HBV-specific immune responses by therapeutic vaccination is a promising, novel treatment option for chronic hepatitis B, it remains unclear whether a loss of HBeAg may influence its efficacy or tolerability. We therefore generated an adeno-associated virus (AAV)-vector that carries a 1.3-fold overlength HBV genome with a typical stop-codon mutation in the pre-core region and initiates the replication of HBeAg(-) HBV in mouse livers. Infection of C57BL/6 mice established persistent HBeAg(-) HBV-replication without any detectable anti-HBV immunity or liver damage. HBV-carrier mice were immunized with , a therapeutic hepatitis B vaccine that uses a particulate HBV S and a core protein for prime vaccination, and a modified vaccinia Ankara (MVA) for boost vaccination. The immunization of HBeAg(+) and HBeAg(-) HBV carrier mice resulted in the effective induction of HBV-specific antibodies and the loss of HBsAg but only mild liver damage. Intrahepatic, HBV-specific CD8 T cells induced in HBeAg(-) mice expressed more IFNγ but showed similar cytolytic activity. This indicates that the loss of HBeAg improves the performance of therapeutic vaccination by enhancing non-cytolytic effector functions.
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http://dx.doi.org/10.3390/vaccines9080841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8402308PMC
July 2021

Comparison of four commercial, automated antigen tests to detect SARS-CoV-2 variants of concern.

Med Microbiol Immunol 2021 Aug 20. Epub 2021 Aug 20.

Max Von Pettenkofer Institute and Gene Center, Virology, National Reference Center for Retroviruses, LMU München, Munich, Germany.

A versatile portfolio of diagnostic tests is essential for the containment of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic. Besides nucleic acid-based test systems and point-of-care (POCT) antigen (Ag) tests, quantitative, laboratory-based nucleocapsid Ag tests for SARS-CoV-2 have recently been launched. Here, we evaluated four commercial Ag tests on automated platforms and one POCT to detect SARS-CoV-2. We evaluated PCR-positive (n = 107) and PCR-negative (n = 303) respiratory swabs from asymptomatic and symptomatic patients at the end of the second pandemic wave in Germany (February-March 2021) as well as clinical isolates EU1 (B.1.117), variant of concern (VOC) Alpha (B.1.1.7) or Beta (B.1.351), which had been expanded in a biosafety level 3 laboratory. The specificities of automated SARS-CoV-2 Ag tests ranged between 97.0 and 99.7% (Lumipulse G SARS-CoV-2 Ag (Fujirebio): 97.03%, Elecsys SARS-CoV-2 Ag (Roche Diagnostics): 97.69%; LIAISON SARS-CoV-2 Ag (Diasorin) and SARS-CoV-2 Ag ELISA (Euroimmun): 99.67%). In this study cohort of hospitalized patients, the clinical sensitivities of tests were low, ranging from 17.76 to 52.34%, and analytical sensitivities ranged from 420,000 to 25,000,000 Geq/ml. In comparison, the detection limit of the Roche Rapid Ag Test (RAT) was 9,300,000 Geq/ml, detecting 23.58% of respiratory samples. Receiver-operating-characteristics (ROCs) and Youden's index analyses were performed to further characterize the assays' overall performance and determine optimal assay cutoffs for sensitivity and specificity. VOCs carrying up to four amino acid mutations in nucleocapsid were detected by all five assays with characteristics comparable to non-VOCs. In summary, automated, quantitative SARS-CoV-2 Ag tests show variable performance and are not necessarily superior to a standard POCT. The efficacy of any alternative testing strategies to complement nucleic acid-based assays must be carefully evaluated by independent laboratories prior to widespread implementation.
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http://dx.doi.org/10.1007/s00430-021-00719-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8377707PMC
August 2021

Prolonged norovirus infections correlate to quasispecies evolution resulting in structural changes of surface-exposed epitopes.

iScience 2021 Jul 30;24(7):102802. Epub 2021 Jun 30.

Institute of Virology, Technische Universität/Helmholtz Zentrum München, 81675 Munich, Germany.

In this study, we analyzed norovirus (NoV) evolution in sequential samples of six chronically infected patients. The capsid gene was amplified from stool samples, and deep sequencing was performed. The role of amino acid flexibility in structural changes and ligand binding was studied with molecular dynamics (MD) simulations. Concentrations of capsid-specific antibodies increased in sequential sera. Capsid sequences accumulated mutations during chronic infection, particularly in the surface-exposed antigenic epitopes A, D, and E. The number of quasispecies increased in infections lasting for >1 month. Interestingly, high genetic complexity and distances were followed by ongoing NoV replication, whereas lower genetic complexity and distances preceded cure. MD simulation revealed that surface-exposed amino acid substitutions of the P2 domain caused fluctuation of blockade epitopes. In conclusion, the capsid protein accumulates numerous mutations during chronic infection; however, only those on the protein surface change the protein structure substantially and may lead to immune escape.
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http://dx.doi.org/10.1016/j.isci.2021.102802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8324856PMC
July 2021

Heterologous prime-boost vaccination with ChAdOx1 nCoV-19 and BNT162b2.

Lancet Infect Dis 2021 09 29;21(9):1212-1213. Epub 2021 Jul 29.

Institute of Virology, School of Medicine, Technical University of Munich/Helmholtz Zentrum München, 81675 Munich, Germany; DZIF, partner sites Munich and Cologne/Bonn, Germany. Electronic address:

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http://dx.doi.org/10.1016/S1473-3099(21)00420-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8321428PMC
September 2021

Intramolecular recombination enables the formation of hepatitis B virus (HBV) cccDNA in mice after HBV genome transfer using recombinant AAV vectors.

Antiviral Res 2021 Oct 17;194:105140. Epub 2021 Jul 17.

Institute of Virology, Technical University of Munich/Helmholtz Zentrum München, Munich, Germany; German Center for Infection Research (DZIF), Munich partner site, Munich, Germany. Electronic address:

The mouse is not a natural host of hepatitis B virus (HBV) infection and - despite engraftment of hepatocytes with the HBV receptor - does not support formation of HBV covalently closed circular (ccc) DNA serving as a template for viral transcription and permitting persistent infection. In a recent study, cccDNA formation in mouse hepatocytes has been described following an HBV genome delivery by a recombinant, adeno-associated virus vector (rAAV) (Lucifora et al., 2017). The integrity of HBV cccDNA, its origin and functionality, however, remained open. In this study, we investigated the identity, origin, and functionality of cccDNA established in mice infected with rAAV carrying 1.3-fold overlength HBV genomes. We show that replication of HBV genotypes A, B, C and D can be initiated in mouse livers, and that cccDNA derived from all genotypes is detected. Restriction enzyme and exonuclease digestion as well as sequencing analysis of cccDNA amplicons revealed authentic HBV cccDNA without any detectable alteration compared to cccDNA established after HBV infection of human liver cells. Mouse livers transduced with a core protein-deficient HBV using rAAV still supported cccDNA formation demonstrating that the genesis of cccDNA was independent of HBV replication. When mice were infected with an rAAV-HBV1.3 carrying premature stop codons in the 5' but not in the 3' core protein open reading frame, the stop codon was partially replaced by the wild-type sequence. This strongly indicated that intramolecular recombination, based on >900 identical base pairs residing at the both ends of the HBV1.3 transgene was the origin of cccDNA formation. Accordingly, we observed a constant loss of cccDNA molecules from mouse livers over time, while HBeAg levels increased over the first two weeks after rAAV-HBV1.3 infection and remained constant thereafter, suggesting a minor contribution of the cccDNA molecules formed to viral transcription and protein expression. In summary, our results provide strong evidence that intramolecular recombination of an overlength, linear HBV genome, but not HBV genome recycling, enables cccDNA formation in rAAV-HBV mouse models.
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http://dx.doi.org/10.1016/j.antiviral.2021.105140DOI Listing
October 2021

Novel function of SART1 in HNF4α transcriptional regulation contributes to its antiviral role during HBV infection.

J Hepatol 2021 Jul 7. Epub 2021 Jul 7.

State Key Laboratory of Virology and Hubei Province Key Laboratory of Allergy and Immunology, Institute of Medical Virology, School of Basic Medical Sciences, Wuhan University, Wuhan, China. Electronic address:

Background & Aims: Our understanding of the interactions between HBV and its host cells is still quite limited. Spliceosome associated factor 1 (SART1) has recently been found to restrict HCV. Thus, we aimed to dissect its role in HBV infection.

Methods: SART1 was knocked down by RNA interference and over-expressed by lentiviral or adeno-associated virus (AAV) vectors in HBV-infected cell cultures and in vivo in HBV-infected mice. Luciferase reporter assays were used to determine viral or host factor promoter activities, and chromatin immunoprecipitation (ChIP) was used to investigate protein-DNA interactions.

Results: In HBV-infected cell cultures, downregulation of SART1 did not affect covalently closed circular HBV DNA but resulted in markedly enhanced HBV RNA, antigen expression and progeny virus production. On the other hand, HBV transcription and replication were significantly inhibited by overexpression of SART1. Similar results were observed in AAV-HBV-infected mice persistently replicating HBV. Inhibition of Janus kinases had no effect on SART1-mediated inhibition of HBV replication. HBV promoter assays revealed that SART1 reduced HBV core promoter activity. By screening known HBV transcription factors, we found that SART1 specifically suppressed the expression of hepatocyte nuclear factor 4α (HNF4α). Luciferase reporter and ChIP assays demonstrated a direct downregulation of HNF4α expression by association of SART1 with the HNF4α proximal P1 promoter element.

Conclusions: We identify SART1 as a novel host factor suppressing HBV cccDNA transcription. Besides its effect on interferon-stimulated genes, SART1 exerts an anti-HBV activity by suppressing HNF4α expression, which is essential for transcription of HBV cccDNA.

Lay Summary: Hepatitis B virus (HBV) infects hepatocytes and persists in the form of covalently closed circular DNA (cccDNA), which remains a major obstacle to successful antiviral treatment. In this study, using various HBV models, we demonstrate that the protein SART1 restricts HBV cccDNA transcription by suppressing a key transcription factor, HNF4α.
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http://dx.doi.org/10.1016/j.jhep.2021.06.038DOI Listing
July 2021

COVID-19 in Patients Receiving CD20-depleting Immunochemotherapy for B-cell Lymphoma.

Hemasphere 2021 Jul 28;5(7):e603. Epub 2021 Jun 28.

Technical University of Munich, School of Medicine, University Hospital Rechts der Isar, Department of Internal Medicine II, Munich, Germany.

The clinical and immunological impact of B-cell depletion in the context of coronavirus disease 2019 (COVID-19) is unclear. We conducted a prospectively planned analysis of COVID-19 in patients who received B-cell depleting anti-CD20 antibodies and chemotherapy for B-cell lymphomas. The control cohort consisted of age- and sex-matched patients without lymphoma who were hospitalized because of COVID-19. We performed detailed clinical analyses, in-depth cellular and molecular immune profiling, and comprehensive virological studies in 12 patients with available biospecimens. B-cell depleted lymphoma patients had more severe and protracted clinical course (median hospitalization 88 versus 17 d). All patients actively receiving immunochemotherapy (n = 5) required ICU support including long-term mechanical ventilation. Neutrophil recovery following granulocyte colony stimulating factor stimulation coincided with hyperinflammation and clinical deterioration in 4 of the 5 patients. Immune cell profiling and gene expression analysis of peripheral blood mononuclear cells revealed early activation of monocytes/macrophages, neutrophils, and the complement system in B-cell depleted lymphoma patients, with subsequent exacerbation of the inflammatory response and dysfunctional interferon signaling at the time of clinical deterioration of COVID-19. Longitudinal immune cell profiling and functional in vitro assays showed SARS-CoV-2-specific CD8 and CD4 T-effector cell responses. Finally, we observed long-term detection of SARS-CoV-2 in respiratory specimens (median 84 versus 12 d) and an inability to mount lasting SARS-CoV-2 antibody responses in B-cell depleted lymphoma patients. In summary, we identified clinically relevant particularities of COVID-19 in lymphoma patients receiving B-cell depleting immunochemotherapies.
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http://dx.doi.org/10.1097/HS9.0000000000000603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8240782PMC
July 2021

T cell engager antibodies enable T cells to control HBV infection and to target HBsAg-positive hepatoma in mice.

J Hepatol 2021 Jun 23. Epub 2021 Jun 23.

Institute of Virology, School of Medicine, Technical University of Munich, Helmholtz Zentrum München, Munich, Germany; German Center for Infection Research (DZIF), Munich and Hamburg Partner sites, Germany. Electronic address:

Background & Aims: Current antiviral therapies control but rarely eliminate HBV, leaving chronic HBV carriers at risk of developing hepatocellular carcinoma (HCC). Lacking or dysfunctional virus-specific adaptive immunity prevents control of HBV and allows the virus to persist. Restoring antiviral T cell immunity could lead to HBV elimination and cure of chronically infected patients.

Methods: We constructed bispecific T cell engager antibodies that are designed to induce antiviral immunity through simultaneous binding of HBV envelope proteins (HBVenv) on infected hepatocytes and CD3 or CD28 on T cells. T cell engager antibodies were employed in co-cultures with healthy donor lymphocytes and HBV-infected target cells. Activation of the T cell response was determined by detection of pro-inflammatory cytokines, effector function (by cytotoxicity) and antiviral effects. To study in vivo efficacy, immune-deficient mice were transplanted with HBVenv-positive and -negative hepatoma cells.

Results: The 2 T cell engager antibodies synergistically activated T cells to become polyfunctional effectors that in turn elicited potent antiviral effects by killing infected cells and in addition controlled HBV via non-cytolytic, cytokine-mediated antiviral mechanisms. In vivo in mice, the antibodies attracted T cells specifically to the tumors expressing HBVenv resulting in T cell activation, tumor infiltration and reduction of tumor burden.

Conclusion: This study demonstrates that the administration of HBVenv-targeting T cell engager antibodies facilitates a robust T cell redirection towards HBV-positive target cells and provides a feasible and promising approach for the treatment of chronic viral hepatitis and HBV-associated HCC.

Lay Summary: T cell engager antibodies are an interesting, novel therapeutic tool to restore immunity in patients with chronic hepatitis B. As bispecific antibodies, they bind envelope proteins on the surface of the hepatitis B virus (HBV) and CD3 or CD28 on T cells. This way, they induce a potent antiviral and cytotoxic T cell response that leads to the elimination of HBV-positive cells. These bispecific T cell engager antibodies are exciting therapeutic candidates for chronic hepatitis B and HBV-associated hepatocellular carcinoma.
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http://dx.doi.org/10.1016/j.jhep.2021.06.022DOI Listing
June 2021

Programmable icosahedral shell system for virus trapping.

Nat Mater 2021 Sep 14;20(9):1281-1289. Epub 2021 Jun 14.

Department of Physics, Technical University of Munich, Munich, Germany.

Broad-spectrum antiviral platforms that can decrease or inhibit viral infection would alleviate many threats to global public health. Nonetheless, effective technologies of this kind are still not available. Here, we describe a programmable icosahedral canvas for the self-assembly of icosahedral shells that have viral trapping and antiviral properties. Programmable triangular building blocks constructed from DNA assemble with high yield into various shell objects with user-defined geometries and apertures. We have created shells with molecular masses ranging from 43 to 925 MDa (8 to 180 subunits) and with internal cavity diameters of up to 280 nm. The shell interior can be functionalized with virus-specific moieties in a modular fashion. We demonstrate this virus-trapping concept by engulfing hepatitis B virus core particles and adeno-associated viruses. We demonstrate the inhibition of hepatitis B virus core interactions with surfaces in vitro and the neutralization of infectious adeno-associated viruses exposed to human cells.
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http://dx.doi.org/10.1038/s41563-021-01020-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611604PMC
September 2021

Hypoxia-Inducible Factor 1 Alpha-Mediated RelB/APOBEC3B Down-regulation Allows Hepatitis B Virus Persistence.

Hepatology 2021 May 15. Epub 2021 May 15.

Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany.

Background And Aims: Therapeutic strategies against HBV focus, among others, on the activation of the immune system to enable the infected host to eliminate HBV. Hypoxia-inducible factor 1 alpha (HIF1α) stabilization has been associated with impaired immune responses. HBV pathogenesis triggers chronic hepatitis-related scaring, leading inter alia to modulation of liver oxygenation and transient immune activation, both factors playing a role in HIF1α stabilization.

Approach And Results: We addressed whether HIF1α interferes with immune-mediated induction of the cytidine deaminase, apolipoprotein B mRNA editing enzyme catalytic subunit 3B (APOBEC3B; A3B), and subsequent covalently closed circular DNA (cccDNA) decay. Liver biopsies of chronic HBV (CHB) patients were analyzed by immunohistochemistry and in situ hybridization. The effect of HIF1α induction/stabilization on differentiated HepaRG or mice ± HBV ± LTβR-agonist (BS1) was assessed in vitro and in vivo. Induction of A3B and subsequent effects were analyzed by RT-qPCR, immunoblotting, chromatin immunoprecipitation, immunocytochemistry, and mass spectrometry. Analyzing CHB highlighted that areas with high HIF1α levels and low A3B expression correlated with high HBcAg, potentially representing a reservoir for HBV survival in immune-active patients. In vitro, HIF1α stabilization strongly impaired A3B expression and anti-HBV effect. Interestingly, HIF1α knockdown was sufficient to rescue the inhibition of A3B up-regulation and -mediated antiviral effects, whereas HIF2α knockdown had no effect. HIF1α stabilization decreased the level of v-rel reticuloendotheliosis viral oncogene homolog B protein, but not its mRNA, which was confirmed in vivo. Noteworthy, this function of HIF1α was independent of its partner, aryl hydrocarbon receptor nuclear translocator.

Conclusions: In conclusion, inhibiting HIF1α expression or stabilization represents an anti-HBV strategy in the context of immune-mediated A3B induction. High HIF1α, mediated by hypoxia or inflammation, offers a reservoir for HBV survival in vivo and should be considered as a restricting factor in the development of immune therapies.
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http://dx.doi.org/10.1002/hep.31902DOI Listing
May 2021

Automated, flow-based chemiluminescence microarray immunoassay for the rapid multiplex detection of IgG antibodies to SARS-CoV-2 in human serum and plasma (CoVRapid CL-MIA).

Anal Bioanal Chem 2021 Sep 13;413(22):5619-5632. Epub 2021 May 13.

Institute of Hydrochemistry, Chair of Analytical Chemistry and Water Chemistry, Technical University of Munich, Elisabeth-Winterhalter-Weg 6, 81377, Munich, Germany.

In the face of the COVID-19 pandemic, the need for rapid serological tests that allow multiplexing emerged, as antibody seropositivity can instruct about individual immunity after an infection with SARS-CoV-2 or after vaccination. As many commercial antibody tests are either time-consuming or tend to produce false negative or false positive results when only one antigen is considered, we developed an automated, flow-based chemiluminescence microarray immunoassay (CL-MIA) that allows for the detection of IgG antibodies to SARS-CoV-2 receptor-binding domain (RBD), spike protein (S1 fragment), and nucleocapsid protein (N) in human serum and plasma in less than 8 min. The CoVRapid CL-MIA was tested with a set of 65 SARS-CoV-2 serology positive or negative samples, resulting in 100% diagnostic specificity and 100% diagnostic sensitivity, thus even outcompeting commercial tests run on the same sample set. Additionally, the prospect of future quantitative assessments (i.e., quantifying the level of antibodies) was demonstrated. Due to the fully automated process, the test can easily be operated in hospitals, medical practices, or vaccination centers, offering a valuable tool for COVID-19 serosurveillance. Graphical abstract.
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http://dx.doi.org/10.1007/s00216-021-03315-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8116441PMC
September 2021

Self-sampling versus health care professional-guided swab collection for SARS-CoV-2 testing.

Infection 2021 May 10. Epub 2021 May 10.

Department of Internal Medicine II, School of Medicine, University Hospital Rechts der Isar, Technical University of Munich, Ismaninger Str. 22, 81675, Munich, Germany.

Purpose: To evaluate the diagnostic reliability and practicability of self-collected oropharyngeal swab samples for the detection of SARS-CoV-2 infection as self-sampling could enable broader testing availability and reduce both personal protective equipment and potential exposure.

Methods: Hospitalized SARS-CoV-2-infected patients were asked to collect two oropharyngeal swabs (SC-OPS1/2), and an additional oropharyngeal swab was collected by a health care professional (HCP-OPS). SARS-CoV-2 PCR testing for samples from 58 participants was performed, with a 48-h delay in half of the self-collected samples (SC-OPS2). The sensitivity, probability of concordance, and interrater reliability were calculated. Univariate and multivariate analyses were performed to assess predictive factors. Practicability was evaluated through a questionnaire.

Results: The test sensitivity for HCP-OPS, SC-OPS1, and SC-OPS2 was 88%, 78%, and 77%, respectively. Combining both SC-OPS results increased the estimated sensitivity to 88%. The concordance probability between HCP-OPS and SC-OPS1 was 77.6% and 82.5% between SC-OPS1 and SC-OPS2, respectively. Of the participants, 69% affirmed performing future self-sampling at home, and 34% preferred self-sampling over HCP-guided testing. Participants with both positive HCP-OPS1 and SC-OPS1 indicating no challenges during self-sampling had more differences in viral load levels between HCP-OPS1 and SC-OPS1 than those who indicated challenges. Increasing disease duration and the presence of anti-SARS-CoV-2-IgG correlated with negative test results in self-collected samples of previously confirmed SARS-CoV-2 positive individuals.

Conclusion: Oropharyngeal self-sampling is an applicable testing approach for SARS-CoV-2 diagnostics. Self-sampling tends to be more effective in early versus late infection and symptom onset, and the collection of two distinct samples is recommended to maintain high test sensitivity.
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http://dx.doi.org/10.1007/s15010-021-01614-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8107404PMC
May 2021

Interferon-induced degradation of the persistent hepatitis B virus cccDNA form depends on ISG20.

EMBO Rep 2021 06 9;22(6):e49568. Epub 2021 May 9.

Institute of Virology, School of Medicine, Technical University of Munich / Helmholtz Zentrum München, Munich, Germany.

Hepatitis B virus (HBV) persists by depositing a covalently closed circular DNA (cccDNA) in the nucleus of infected cells that cannot be targeted by available antivirals. Interferons can diminish HBV cccDNA via APOBEC3-mediated deamination. Here, we show that overexpression of APOBEC3A alone is not sufficient to reduce HBV cccDNA that requires additional treatment of cells with interferon indicating involvement of an interferon-stimulated gene (ISG) in cccDNA degradation. Transcriptome analyses identify ISG20 as the only type I and II interferon-induced, nuclear protein with annotated nuclease activity. ISG20 localizes to nucleoli of interferon-stimulated hepatocytes and is enriched on deoxyuridine-containing single-stranded DNA that mimics transcriptionally active, APOBEC3A-deaminated HBV DNA. ISG20 expression is detected in human livers in acute, self-limiting but not in chronic hepatitis B. ISG20 depletion mitigates the interferon-induced loss of cccDNA, and co-expression with APOBEC3A is sufficient to diminish cccDNA. In conclusion, non-cytolytic HBV cccDNA decline requires the concerted action of a deaminase and a nuclease. Our findings highlight that ISGs may cooperate in their antiviral activity that may be explored for therapeutic targeting.
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http://dx.doi.org/10.15252/embr.201949568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8183418PMC
June 2021

Molecular regulation of the hepatic bile acid uptake transporter and HBV entry receptor NTCP.

Biochim Biophys Acta Mol Cell Biol Lipids 2021 08 29;1866(8):158960. Epub 2021 Apr 29.

Tytgat Institute for Liver and Intestinal Research, Amsterdam University Medical Centers Amsterdam, the Netherlands; Amsterdam Gastroenterology, Endocrinology, Metabolism (AGEM), Amsterdam, the Netherlands; Department of Gastroenterology and Hepatology, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, the Netherlands. Electronic address:

Transporters expressed by hepatocytes and enterocytes play a critical role in maintaining the enterohepatic circulation of bile acids. The sodium taurocholate cotransporting polypeptide (NTCP), exclusively expressed at the basolateral side of hepatocytes, mediates the uptake of conjugated bile acids. In conditions where bile flow is impaired (cholestasis), pharmacological inhibition of NTCP-mediated bile acid influx is suggested to reduce hepatocellular damage due to bile acid overload. Furthermore, NTCP has been shown to play an important role in hepatitis B virus (HBV) and hepatitis Delta virus (HDV) infection by functioning as receptor for viral entry into hepatocytes. This review provides a summary of current molecular insight into the regulation of NTCP expression at the plasma membrane, hepatic bile acid transport, and NTCP-mediated viral infection.
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http://dx.doi.org/10.1016/j.bbalip.2021.158960DOI Listing
August 2021

SARS-CoV-2 serology increases diagnostic accuracy in CT-suspected, PCR-negative COVID-19 patients during pandemic.

Respir Res 2021 Apr 23;22(1):119. Epub 2021 Apr 23.

Department of Internal Medicine II, School of Medicine, Technical University of Munich, Munich, Germany.

Background: In the absence of PCR detection of SARS-CoV-2 RNA, accurate diagnosis of COVID-19 is challenging. Low-dose computed tomography (CT) detects pulmonary infiltrates with high sensitivity, but findings may be non-specific. This study assesses the diagnostic value of SARS-CoV-2 serology for patients with distinct CT features but negative PCR.

Methods: IgM/IgG chemiluminescent immunoassay was performed for 107 patients with confirmed (group A: PCR + ; CT ±) and 46 patients with suspected (group B: repetitive PCR-; CT +) COVID-19, admitted to a German university hospital during the pandemic's first wave. A standardized, in-house CT classification of radiological signs of a viral pneumonia was used to assess the probability of COVID-19.

Results: Seroconversion rates (SR) determined on day 5, 10, 15, 20 and 25 after symptom onset (SO) were 8%, 25%, 65%, 76% and 91% for group A, and 0%, 10%, 19%, 37% and 46% for group B, respectively; (p < 0.01). Compared to hospitalized patients with a non-complicated course (non-ICU patients), seroconversion tended to occur at lower frequency and delayed in patients on intensive care units. SR of patients with CT findings classified as high certainty for COVID-19 were 8%, 22%, 68%, 79% and 93% in group A, compared with 0%, 15%, 28%, 50% and 50% in group B (p < 0.01). SARS-CoV-2 serology established a definite diagnosis in 12/46 group B patients. In 88% (8/9) of patients with negative serology > 14 days after symptom onset (group B), clinico-radiological consensus reassessment revealed probable diagnoses other than COVID-19. Sensitivity of SARS-CoV-2 serology was superior to PCR > 17d after symptom onset.

Conclusions: Approximately one-third of patients with distinct COVID-19 CT findings are tested negative for SARS-CoV-2 RNA by PCR rendering correct diagnosis difficult. Implementation of SARS-CoV-2 serology testing alongside current CT/PCR-based diagnostic algorithms improves discrimination between COVID-19-related and non-related pulmonary infiltrates in PCR negative patients. However, sensitivity of SARS-CoV-2 serology strongly depends on the time of testing and becomes superior to PCR after the 2 week following symptom onset.
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http://dx.doi.org/10.1186/s12931-021-01717-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8062836PMC
April 2021

Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV.

Nature 2021 06 12;594(7862):246-252. Epub 2021 Apr 12.

Technical University of Munich, School of Medicine, Institute of Virology, Munich, Germany.

The emergence and global spread of SARS-CoV-2 has resulted in the urgent need for an in-depth understanding of molecular functions of viral proteins and their interactions with the host proteome. Several individual omics studies have extended our knowledge of COVID-19 pathophysiology. Integration of such datasets to obtain a holistic view of virus-host interactions and to define the pathogenic properties of SARS-CoV-2 is limited by the heterogeneity of the experimental systems. Here we report a concurrent multi-omics study of SARS-CoV-2 and SARS-CoV. Using state-of-the-art proteomics, we profiled the interactomes of both viruses, as well as their influence on the transcriptome, proteome, ubiquitinome and phosphoproteome of a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed crosstalk between the perturbations taking place upon infection with SARS-CoV-2 and SARS-CoV at different levels and enabled identification of distinct and common molecular mechanisms of these closely related coronaviruses. The TGF-β pathway, known for its involvement in tissue fibrosis, was specifically dysregulated by SARS-CoV-2 ORF8 and autophagy was specifically dysregulated by SARS-CoV-2 ORF3. The extensive dataset (available at https://covinet.innatelab.org ) highlights many hotspots that could be targeted by existing drugs and may be used to guide rational design of virus- and host-directed therapies, which we exemplify by identifying inhibitors of kinases and matrix metalloproteases with potent antiviral effects against SARS-CoV-2.
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http://dx.doi.org/10.1038/s41586-021-03493-4DOI Listing
June 2021

Increased HERV-K(HML-2) Transcript Levels Correlate with Clinical Parameters of Liver Damage in Hepatitis C Patients.

Cells 2021 Mar 31;10(4). Epub 2021 Mar 31.

Institute of Virology, HelmholtzZentrum München, Neuherberg 85764, Germany.

Chronic hepatitis C virus (HCV) infection is closely associated with a plethora of diseases, including cancers and autoimmune disorders. However, the distinct triggers and cellular networks leading to such HCV-derived diseases are poorly understood. Around 8% of the human genome consists of human endogenous retroviruses. They are usually silenced but can be reactivated by environmental conditions, including viral infections. Our current understanding indicates that the activation of one specific family-namely, HERV-K(HML-2)-is linked to distinct pathologies, including cancer and autoimmunity. In this study, we analyzed the transcription levels of HERV-K(HML-2) in 42 HCV-infected patients receiving direct-acting antiviral therapies. Samples from the start of treatment until 12 weeks post-treatment were investigated. Our results show increased HERV-K(HML-2) transcript levels in patients with HCV-derived liver cirrhosis throughout the observation period. Several clinical parameters specifying poor liver function are positively correlated with HERV-K(HML-2) expression. Of note, patients without a sustained viral clearance showed a drastic increase in HERV-K(HML-2) transcript levels. Together, our data suggest that increased HERV-K(HML-2) expression is correlated with reduced liver function as well as therapy success in HCV-infected patients.
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http://dx.doi.org/10.3390/cells10040774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8065411PMC
March 2021

Fetomaternal immune cross talk modifies T-cell priming through sustained changes to DC function.

J Allergy Clin Immunol 2021 Sep 5;148(3):843-857.e6. Epub 2021 Mar 5.

Institute for Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Munich, Germany. Electronic address:

Background: Prenatal exposure to infections can modify immune development. These environmental disturbances during early life potentially alter the incidence of inflammatory disorders as well as priming of immune responses. Infection with the helminth Schistosoma mansoni is widely studied for its ability to alter immune responsiveness and is associated with variations in coinfection, allergy, and vaccine efficacy in endemic populations.

Objective: Exposure to maternal schistosomiasis during early life, even without transmission of infection, can result in priming effects on offspring immune responses to bystander antigenic challenges as related to allergic responsiveness and vaccination, with this article seeking to further clarify the effects and underlying immunologic imprinting.

Methods: Here, we have combined a model of chronic maternal schistosomiasis infection with a thorough analysis of subsequent offspring immune responses to allergy and vaccination models, including viral challenge and steady-state changes to immune cell compartments.

Results: We have demonstrated that maternal schistosomiasis alters CD4 responses during allergic sensitization and challenge in a skewed IL-4/B-cell-dominant response to antigenic challenge associated with limited inflammatory response. Beyond that, we have uncovered previously unidentified alterations to CD8 T-cell responses during immunization that are dependent on vaccine formulation and have functional impact on the efficacy of vaccination against viral infection in a murine hepatitis B virus model.

Conclusion: In addition to steady-state modifications to CD4 T-cell polarization and B-cell priming, we have traced these modified CD8 responses to an altered dendritic cell phenotype sustained into adulthood, providing evidence for complex priming effects imparted by infection via fetomaternal cross talk.
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http://dx.doi.org/10.1016/j.jaci.2021.02.031DOI Listing
September 2021

Mucosal-Associated Invariant T (MAIT) Cells Are Highly Activated and Functionally Impaired in COVID-19 Patients.

Viruses 2021 02 3;13(2). Epub 2021 Feb 3.

Department of Internal Medicine II, University Hospital Rechts der Isar, School of Medicine, Technical University of Munich (TUM), 81675 Munich, Germany.

Coronavirus disease 2019 (COVID-19), caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprises mild courses of disease as well as progression to severe disease, characterised by lung and other organ failure. The immune system is considered to play a crucial role for the pathogenesis of COVID-19, although especially the contribution of innate-like T cells remains poorly understood. Here, we analysed the phenotype and function of mucosal-associated invariant T (MAIT) cells, innate-like T cells with potent antimicrobial effector function, in patients with mild and severe COVID-19 by multicolour flow cytometry. Our data indicate that MAIT cells are highly activated in patients with COVID-19, irrespective of the course of disease, and express high levels of proinflammatory cytokines such as IL-17A and TNFα ex vivo. Of note, expression of the activation marker HLA-DR positively correlated with SAPS II score, a measure of disease severity. Upon MAIT cell-specific in vitro stimulation, MAIT cells however failed to upregulate expression of the cytokines IL-17A and TNFα, as well as cytolytic proteins, that is, granzyme B and perforin. Thus, our data point towards an altered cytokine expression profile alongside an impaired antibacterial and antiviral function of MAIT cells in COVID-19 and thereby contribute to the understanding of COVID-19 immunopathogenesis.
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http://dx.doi.org/10.3390/v13020241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7913667PMC
February 2021

Hypoxia inducible factors regulate hepatitis B virus replication by activating the basal core promoter.

J Hepatol 2021 Jul 29;75(1):64-73. Epub 2021 Jan 29.

Nuffield Department of Medicine, University of Oxford, Oxford, UK; Chinese Academy of Medical Sciences (CAMS) Oxford Institute (COI), University of Oxford, Oxford, UK. Electronic address:

Background & Aims: Hypoxia inducible factors (HIFs) are a hallmark of inflammation and are key regulators of hepatic immunity and metabolism, yet their role in HBV replication is poorly defined. HBV replicates in hepatocytes within the liver, a naturally hypoxic organ, however most studies of viral replication are performed under conditions of atmospheric oxygen, where HIFs are inactive. We therefore investigated the role of HIFs in regulating HBV replication.

Methods: Using cell culture, animal models, human tissue and pharmacological agents inhibiting the HIF-prolyl hydroxylases, we investigated the impact of hypoxia on the HBV life cycle.

Results: Culturing liver cell-based model systems under low oxygen uncovered a new role for HIFs in binding HBV DNA and activating the basal core promoter, leading to increased pre-genomic RNA and de novo HBV particle secretion. The presence of hypoxia responsive elements among all primate members of the hepadnaviridae highlights an evolutionary conserved role for HIFs in regulating this virus family.

Conclusions: Identifying a role for this conserved oxygen sensor in regulating HBV transcription suggests that this virus has evolved to exploit the HIF signaling pathway to persist in the low oxygen environment of the liver. Our studies show the importance of considering oxygen availability when studying HBV-host interactions and provide innovative routes to better understand and target chronic HBV infection.

Lay Summary: Viral replication in host cells is defined by the cellular microenvironment and one key factor is local oxygen tension. Hepatitis B virus (HBV) replicates in the liver, a naturally hypoxic organ. Hypoxia inducible factors (HIFs) are the major sensors of low oxygen; herein, we identify a new role for these factors in regulating HBV replication, revealing new therapeutic targets.
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http://dx.doi.org/10.1016/j.jhep.2020.12.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8214165PMC
July 2021

Linear B-Cell Epitopes in Human Norovirus GII.4 Capsid Protein Elicit Blockade Antibodies.

Vaccines (Basel) 2021 Jan 14;9(1). Epub 2021 Jan 14.

Institute of Virology, School of Medicine, Technical University of Munich, 81675 Munich, Germany.

Human norovirus (HuNoV) is the leading cause of nonbacterial gastroenteritis worldwide with the GII.4 genotype accounting for over 80% of infections. The major capsid protein of GII.4 variants is evolving rapidly, resulting in new epidemic variants with altered antigenic potentials that must be considered for the development of an effective vaccine. In this study, we identify and characterize linear blockade B-cell epitopes in HuNoV GII.4. Five unique linear B-cell epitopes, namely P2A, P2B, P2C, P2D, and P2E, were predicted on the surface-exposed regions of the capsid protein. Evolving of the surface-exposed epitopes over time was found to correlate with the emergence of new GII.4 outbreak variants. Molecular dynamic simulation (MD) analysis and molecular docking revealed that amino acid substitutions in the putative epitopes P2B, P2C, and P2D could be associated with immune escape and the appearance of new GII.4 variants by affecting solvent accessibility and flexibility of the antigenic sites and histo-blood group antigens (HBAG) binding. Testing the synthetic peptides in wild-type mice, epitopes P2B (336-355), P2C (367-384), and P2D (390-400) were recognized as GII.4-specific linear blockade epitopes with the blocking rate of 68, 55 and 28%, respectively. Blocking rate was found to increase to 80% using the pooled serum of epitopes P2B and P2C. These data provide a strategy for expanding the broad blockade potential of vaccines for prevention of NoV infection.
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http://dx.doi.org/10.3390/vaccines9010052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830539PMC
January 2021

Evaluation of two rapid antigen tests to detect SARS-CoV-2 in a hospital setting.

Med Microbiol Immunol 2021 Feb 16;210(1):65-72. Epub 2021 Jan 16.

Max von Pettenkofer Institute and Gene Center, Virology, National Reference Center for Retroviruses, LMU München, Pettenkoferstr. 9a, 80336, Munich, Germany.

Successful containment strategies for the SARS-CoV-2 pandemic will depend on reliable diagnostic assays. Point-of-care antigen tests (POCT) may provide an alternative to time-consuming PCR tests to rapidly screen for acute infections on site. Here, we evaluated two SARS-CoV-2 antigen tests: the STANDARD™ F COVID-19 Ag FIA (FIA) and the SARS-CoV-2 Rapid Antigen Test (RAT). For diagnostic assessment, we used a large set of PCR-positive and PCR-negative respiratory swabs from asymptomatic and symptomatic patients and health care workers in the setting of two University Hospitals in Munich, Germany, i.e. emergency rooms, patient care units or employee test centers. For FIA, overall clinical sensitivity and specificity were 45.4% (n = 381) and 97.8% (n = 360), respectively, and for RAT, 50.3% (n = 445) and 97.7% (n = 386), respectively. For primary diagnosis of asymptomatic and symptomatic individuals, diagnostic sensitivities were 60.9% (FIA) (n = 189) and 64.5% (RAT) (n = 256). This questions these tests' utility for the reliable detection of acute SARS-CoV-2-infected individuals, in particular in high-risk settings. We support the proposal that convincing high-quality outcome data on the impact of false-negative and false-positive antigen test results need to be obtained in a POCT setting. Moreover, the efficacy of alternative testing strategies to complement PCR assays must be evaluated by independent laboratories, prior to widespread implementation in national and international test strategies.
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http://dx.doi.org/10.1007/s00430-020-00698-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7811156PMC
February 2021

Efficient and reproducible depletion of hepatitis B virus from plasma derived extracellular vesicles.

J Extracell Vesicles 2020 12 22;10(2):e12040. Epub 2020 Dec 22.

Institute of Virology School of Medicine Helmholtz Zentrum München/Technical University of Munich Garching Germany.

Extracellular vesicles (EVs) are emerging fundamental players in viral infections by shuttling viral components, mediating immune responses and likely the spread of the virus. However, the obstacles involved in purifying EVs and removing contaminating viral particles in a reliable and effective manner bottlenecks the full potential for the development of clinical and diagnostic treatment options targeting EV. Because of the similarities in size, density, membrane composition and mode of biogenesis of EVs and virions there are no standardized approaches for virus-removal from EV preparations yet. Functional EV studies also require EV samples that are devoid of antibody contaminants. Consequently, the study of EVs in virology needs reliable and effective protocols to purify EVs and remove contaminating antibodies and viral particles. Here, we established a protocol for EV purification from hepatitis B virus (HBV)-containing plasma by a combination of size-exclusion chromatography and affinity-based purification. After purification, EV samples were free of virus-sized particles, HBV surface antigen, HBV core antigen, antibodies or infectious material. Viral genomic contamination was also decreased following purification. By using appropriate antibodies and size parameters, this protocol could potentially be applied to purification of EVs from other viral samples. In summary, we established a fast, reproducible and robust approach for the removal of HBV from EV preparations. Looking forward to the point of purifying EVs from clinical samples, this method should enable studies shedding light on the underlying mechanisms of EVs in viral infections and their diagnostic and prognostic potential.
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http://dx.doi.org/10.1002/jev2.12040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7754750PMC
December 2020

Global Occurrence of Clinically Relevant Hepatitis B Virus Variants as Found by Analysis of Publicly Available Sequencing Data.

Viruses 2020 11 23;12(11). Epub 2020 Nov 23.

Institute of Virology, Technical University of Munich/Helmholtz Zentrum München, Trogerstrasse 30, D-81675 München, Germany.

Several viral factors impact the natural course of hepatitis B virus (HBV) infection, the sensitivity of diagnostic tests, or treatment response to interferon-α and nucleos(t)ide analogues. These factors include the viral genotype and serotype but also mutations affecting the HBV surface antigen, basal core promoter/pre-core region, or reverse transcriptase. However, a comprehensive overview of the distribution of HBV variants between HBV genotypes or different geographical locations is lacking. To address this, we performed an in silico analysis of publicly available HBV full-length genome sequences. We found that not only the serotype frequency but also the majority of clinically relevant mutations are primarily associated with specific genotypes. Distinct mutations enriched in certain world regions are not explained by the local genotype distribution. Two HBV variants previously identified to confer resistance to the nucleotide analogue tenofovir in vitro were not identified, questioning their translational relevance. In summary, our work elucidates the differences in the clinical manifestation of HBV infection observed between genotypes and geographical locations and furthermore helps identify suitable diagnostic tests and therapies.
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http://dx.doi.org/10.3390/v12111344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7700573PMC
November 2020

Reply to the Letter of Charre et al. "Mis-Genotyping of Some Hepatitis D Virus Genotype 2 and 5 Sequences Using HDVdb".

Viruses 2020 11 9;12(11). Epub 2020 Nov 9.

Institute of Virology, Technische Universität München, 81675 Munich, Germany.

We thank Charre and colleagues for spotting the mis-annotation of sequences in our database, which was caused by human error [...].
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http://dx.doi.org/10.3390/v12111278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7698116PMC
November 2020
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