Publications by authors named "Nicole Fischer"

105 Publications

Recommendations for the introduction of metagenomic next-generation sequencing in clinical virology, part II: bioinformatic analysis and reporting.

J Clin Virol 2021 Mar 26;138:104812. Epub 2021 Mar 26.

Virology Laboratory, Genomics and Health Area, Centre for Public Health Research (FISABIO-Public Health), Valencia, Spain; Department of Microbiology, Medical School, University of Valencia, Spain; CIBERESP, Instituto de Salud Carlos III, Madrid, Spain. Electronic address:

Metagenomic next-generation sequencing (mNGS) is an untargeted technique for determination of microbial DNA/RNA sequences in a variety of sample types from patients with infectious syndromes. mNGS is still in its early stages of broader translation into clinical applications. To further support the development, implementation, optimization and standardization of mNGS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mNGS for viral diagnostics to share methodologies and experiences, and to develop application guidelines. Following the ENNGS publication Recommendations for the introduction of mNGS in clinical virology, part I: wet lab procedure in this journal, the current manuscript aims to provide practical recommendations for the bioinformatic analysis of mNGS data and reporting of results to clinicians.
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http://dx.doi.org/10.1016/j.jcv.2021.104812DOI Listing
March 2021

Validation of the German Version of the Mind Excessively Wandering Scale (MEWS-G).

Fortschr Neurol Psychiatr 2021 Feb 26. Epub 2021 Feb 26.

Klinik für Psychiatrie und Psychotherapie, Zentralinstitut für Seelische Gesundheit Klinische Fakultät Mannheim, Universität Heidelberg.

Increasing evidence shows that unintentional mind wandering is linked to Attention Deficit Hyperactivity Disorder (ADHD) and that its frequency contributes to symptom severity and functional impairment in ADHD. However, empirical data on mind wandering in adult ADHD are still scarce, and a validated scale to assess mind wandering in German adult ADHD patients is lacking. The primary aim of this study is to assess the psychometric properties of the German version of the recently published Mind Excessively Wandering Scale (MEWS-G) in terms of factorial structure and factor stability, internal consistency and construct validity. Analyses were performed in 128 adults with ADHD, clinical and healthy controls. As described for the original English 15-item version of the scale, we found lowest item-total-correlations for items 6, 10 and 14 with item-total correlation of all: 0.54/ADHD: 0.32 (item 6), all: 0.55/ADHD: 0.39 (item 10) and all: 0.11/ADHD: -0.04 (item 14). Item-total correlations for the remaining items were 0.65-0.86 and Cronbach Alpha was 0.96 indicating good internal consistency of the 12-item version of scale, on which we based all further analyses. Principal component analysis indicated a one- and two- factorial scale structure respectively explaining 71.7 % and 78.7 % of variance. Both factors showed good stability with lower stability of the factor-2 solution if sample size was reduced. The two-factorial solution also had many cross-loadings and a strong correlation of both factors in confirmatory factorial analysis (rf1f2 = 0.87). It probably describes related and interdependent, but not distinct facets of mind wandering, which strongly argues for the one factorial structure of the scale. Mean MEWS-G score in ADHD was 23.77 ± 7.85 compared to 7.64 ± 7.27 in controls (p < .0001). According to ROC, the optimal cut-off point to discriminate ADHD and controls is at MEWS-G score = 13. On the symptom level, MEWS-G score was correlated with ADHD, depressive and total psychiatric symptom scores, on the personality level with neuroticsm and negatively with conscientiousness and on the functional level with social interaction difficulties and impaired self-efficacy. In summary, our study shows that MEWS-G is a reliable, valid instrument to assess spontaneous mind wandering in adult ADHD and to discriminate between ADHD and controls.
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http://dx.doi.org/10.1055/a-1362-9743DOI Listing
February 2021

Clinical evaluation of a laboratory-developed quantitative BK virus-PCR assay using the cobas® omni Utility Channel.

J Virol Methods 2021 Apr 4;290:114093. Epub 2021 Feb 4.

Department of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Background: In immunocompromised patients, BK Virus (BKV) reactivation may cause serious disease with high morbidity. Particularly for patient management after solid organ transplantation, monitoring of viral load in different clinical specimens is crucial to ensure early diagnosis and response to reactivation. In this study, we evaluated the clinical performance of a custom designed primer /probe set for detection of BKV on the cobas® 6800, a high-throughput platform, employing the open channel of the system for integration of a lab-developed test (LDT).

Materials/methods: A primer/probe set was optimized for the use on a high-throughput platform. Clinical performance was assessed in EDTA-plasma, serum and urine samples. Limit-of-detection (LOD) was determined by using a dilution series of BKV WHO standard. A CE-labeled PCR test (Altona Diagnostics) was used as a comparison to the assay.

Results: The LOD for the LDT BKV assay was 6.7 IU/mL. Inter-and intra-run variability (at 5 x LOD) was low (<1.5 Ct in all specimens). All quality control panel specimens (Instand Germany n = 19) were correctly identified. Of 290 clinical samples tested, results were concordant for 280 samples. Sensitivity and specificity of the assay were 96 % and 98 % respectively. The quantitative analysis revealed a strong correlation (linear regression) between the CE-labelled comparator assay and the new BKV LDT assay with r = 0.96 for n = 123 urine samples and r = 0.98 for n = 167 plasma/serum samples.

Conclusion: Compared to a CE-IVD assay, the adapted LDT showed good analytical and clinical sensitivity and specificity for the detection and quantification of BKV in different clinical specimens. It represents a convenient solution to automate the LDT workflow with low hands-on time and thus facilitates high-throughput screening for BKV reactivation in immunocompromised patients.
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http://dx.doi.org/10.1016/j.jviromet.2021.114093DOI Listing
April 2021

Perspectives of 281 patients with Mayer-Rokitansky-Küster-Hauser Syndrome on uterine transplantation.

Fertil Steril 2021 Apr 16;115(4):1074-1083. Epub 2021 Jan 16.

Department of Obstetrics and Gynecology, Division of Reproductive Sciences & Women's Health Research, Johns Hopkins Hospital, Baltimore, Maryland. Electronic address:

Objectives: To investigate the personal, ethical, and financial perspectives of individuals with Mayer-Rokitansky-Küster-Hauser syndrome (MRKH), a congenital uterine factor infertility condition, regarding uterine transplantation (UTx).

Design: Cross-sectional, quantitative survey.

Setting: A 60-item anonymous electronic questionnaire was disseminated via social media sites.

Patients: International members of the Beautiful You MRKH Foundation.

Interventions: None.

Main Outcome Measures: The survey contained UTx educational materials followed by questions assessing participants' baseline knowledge, global perceptions, financial concerns, and ethical considerations regarding UTx.

Results: We received 281 responses, with a mean participant age of 28.2 ± 9.8 years. After reviewing the education material, most participants considered receiving a UTx (73%), believed that it should be an option for all women with uterine factor infertility (86%), and believed that it should be covered by health insurance (78%). Respondents perceived the benefits of the procedure to outweigh the risks (67%) and considered it to be an ethical procedure (82%). Almost one-half (49%) were willing to spend more than $10,000 out of pocket to receive the procedure. When asked to rank the risk of UTx to self, donor, and fetus in order of personal importance, 21% ranked their own safety last.

Conclusion: There is a profound desire in the MRKH community for UTx to become more widely available and affordable. MRKH patients may represent a vulnerable population requiring special considerations for informed consent and rigorous evaluation for UTx. Providers caring for MRKH patients should be prepared to provide education about UTx and to thoughtfully engage with news and media outlets to communicate evidence-supported information.
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http://dx.doi.org/10.1016/j.fertnstert.2020.10.044DOI Listing
April 2021

Brainstem Pathologies Correlate With Depression and Psychosis in Parkinson's Disease.

Am J Geriatr Psychiatry 2021 Jan 14. Epub 2021 Jan 14.

Department of Psychiatry and Behavioral Sciences (NMF, JTH, KP, MDS, AB, GMP), Johns Hopkins School of Medicine, Baltimore, MD; Morris K. Udall Parkinson's Disease Research Center of Excellence (CCB, ZM, KAM, LSR, AP, JCT, GMP), Johns Hopkins School of Medicine, Baltimore, MD; Department of Neurology (AB, TMD, CLM, KAM, LSR, AP, GMP), Johns Hopkins School of Medicine, Baltimore MD. Electronic address:

Background: The pathological hallmarks of Parkinson's disease include intraneuronal Lewy bodies, neuronal loss, and gliosis. We aim to correlate Parkinson's disease neuropsychiatric symptoms, (e.g., depression, psychosis, and anxiety) with the severity of neuropathology in the substantia nigra and locus coeruleus.

Methods: The brains of 175 participants with a primary pathologic diagnosis of Parkinson's disease were analyzed semi-quantitatively to ascertain the burden of neuronal loss and gliosis and Lewy body pathology within the locus coeruleus and substantia nigra. Participants' history of anxiety, depression, and psychosis were determined using a chart-extracted medical history or record of formal psychiatric evaluation.

Results: Of the sample, 56% (n = 98), 50% (n = 88), and 31.25% (n = 55) of subjects had a diagnosis of psychosis, depression, and anxiety, respectively. Psychosis (χ = 7.1, p = 0.008, df = 1) and depression (χ = 7.2, p = 0.007, df = 1) were associated with severe neuronal loss and gliosis in the substantia nigra but not in the locus coeruleus. No association was observed between anxiety and neuronal loss and gliosis in either region. No neuropsychiatric symptoms were associated with Lewy body score. After controlling for disease duration and dementia, psychosis (odds ratio [OR]: 3.1, 95% confidence interval [CI]: 1.5-6.4, χ = 9.4, p = 0.012, df = 1) and depression (OR: 2.6, 95% CI: 1.3-5.0, χ = 7.9, p = 0.005, df = 1) remained associated with severe neuronal loss and gliosis in the substantia nigra.

Conclusion: These results suggest that psychosis and depression in Parkinson's disease are associated with the underlying neurodegenerative process and demonstrate that cell loss and gliosis may be a better marker of neuropsychiatric symptoms than Lewy body pathology.
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http://dx.doi.org/10.1016/j.jagp.2020.12.009DOI Listing
January 2021

Protecting Our Youth: Support Policy to Combat Health Disparities Fueled by Targeted Food Advertising.

J Am Heart Assoc 2021 Jan 30;10(1):e018900. Epub 2020 Dec 30.

Ciccarone Center for the Prevention of Cardiovascular Disease Division of Cardiology Johns Hopkins University School of Medicine Baltimore MD.

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http://dx.doi.org/10.1161/JAHA.120.018900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955455PMC
January 2021

Upregulation of HLA-F expression by BK polyomavirus infection induces immune recognition by KIR3DS1-positive natural killer cells.

Kidney Int 2020 Dec 23. Epub 2020 Dec 23.

German Center for Infection Research (DZIF), Partner site Hamburg-Lübeck-Borstel-Riems, Hamburg, Germany; Research Department Virus Immunology, Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany. Electronic address:

BK polyomavirus-associated nephropathy is a common complication after kidney transplantation leading to reduced graft function or loss. The molecular pathogenesis of BK polyomavirus-induced nephropathy is not well understood. A recent study had described a protective effect of the activating natural killer cell receptor KIR3DS1 in BK polyomavirus-associated nephropathy, suggesting a role of NK cells in modulating disease progression. Using an in vitro cell culture model of human BK polyomavirus infection and kidney biopsy samples from patients with BK polyomavirus-associated nephropathy, we observed significantly increased surface expression of the ligand for KIR3DS1, HLA-F, on BK polyomavirus-infected kidney tubular cells. Upregulation of HLA-F expression resulted in significantly increased binding of KIR3DS1 to BK polyomavirus-infected cells and activation of primary KIR3DS-positive natural killer cells. Thus, our data provide a mechanism by which KIR3DS-positive natural killer cells can control BK polyomavirus infection of the kidney, and rationale for exploring HLA-F/KIR3DS1 interactions for immunotherapeutic approaches in BK polyomavirus-associated nephropathy.
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http://dx.doi.org/10.1016/j.kint.2020.12.014DOI Listing
December 2020

Merkel Cell Polyomavirus Encodes Circular RNAs (circRNAs) Enabling a Dynamic circRNA/microRNA/mRNA Regulatory Network.

mBio 2020 12 15;11(6). Epub 2020 Dec 15.

Hillman Cancer Center, Cancer Virology Program, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

Viral noncoding RNAs have acquired increasing prominence as important regulators of infection and mediators of pathogenesis. Circular RNAs (circRNAs) generated by backsplicing events have been identified in several oncogenic human DNA viruses. Here, we show that Merkel cell polyomavirus (MCV), the etiologic cause of ∼80% of Merkel cell carcinomas (MCCs), also expresses circular RNAs. By RNase R-resistant RNA sequencing, four putative circRNA backsplice junctions (BSJs) were identified from the MCV early region (ER). The most abundantly expressed MCV circRNA, designated circMCV-T, is generated through backsplicing of all of ER exon II to form a 762-nucleotide (nt) circular RNA molecule. Curiously, circMCV-T, as well as two other less abundantly expressed putative MCV circRNAs, overlaps in a complementary fashion with the MCV microRNA (miRNA) locus that encodes MCV-miR-M1. circMCV-T is consistently detected in concert with linear T antigen transcripts throughout infection, suggesting a crucial role for this RNA molecule in the regulatory functions of the early region, known to be vital for viral replication. Knocking out the hairpin structure of MCV-miR-M1 in genomic early region expression constructs and using a new high-efficiency, recombinase-mediated, recircularized MCV molecular clone demonstrates that circMCV-T levels decrease in the presence of MCV-miR-M1, underscoring the interplay between MCV circRNA and miRNA. Furthermore, circMCV-T partially reverses the known inhibitory effect of MCV-miR-M1 on early gene expression. RNase R-resistant RNA sequencing of lytic rat polyomavirus 2 (RatPyV2) identified an analogously located circRNA, stipulating a crucial, conserved regulatory function of this class of RNA molecules in the family of polyomaviruses. Covalently closed circular RNAs were recently described in the human DNA tumor viruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and human papillomavirus (HPV). Here, we show that MCV, another DNA tumor virus, generates circRNAs from its early regulatory region in concert with T antigen linear transcripts. MCV circMCV-T interacts with another MCV noncoding RNA, miR-M1, to functionally modulate early region transcript expression important for viral replication and long-term episomal persistence. This work describes a dynamic regulatory network integrating circRNA/miRNA/mRNA biomolecules and underscores the intricate functional modulation between several classes of polyomavirus-encoded RNAs in the control of viral replication.
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http://dx.doi.org/10.1128/mBio.03059-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773998PMC
December 2020

Recommendations for the introduction of metagenomic high-throughput sequencing in clinical virology, part I: Wet lab procedure.

J Clin Virol 2021 Jan 18;134:104691. Epub 2020 Nov 18.

Department of Medical Microbiology, Leiden University Medical Center, Leiden, the Netherlands. Electronic address:

Metagenomic high-throughput sequencing (mHTS) is a hypothesis-free, universal pathogen detection technique for determination of the DNA/RNA sequences in a variety of sample types and infectious syndromes. mHTS is still in its early stages of translating into clinical application. To support the development, implementation and standardization of mHTS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mHTS for viral diagnostics to share methodologies and experiences, and to develop application recommendations. This manuscript aims to provide practical recommendations for the wet lab procedures necessary for implementation of mHTS for virus diagnostics and to give recommendations for development and validation of laboratory methods, including mHTS quality assurance, control and quality assessment protocols.
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http://dx.doi.org/10.1016/j.jcv.2020.104691DOI Listing
January 2021

Angiotensin-Converting Enzyme Inhibitors Reduce Uterine Fibroid Incidence in Hypertensive Women.

J Clin Endocrinol Metab 2021 Jan;106(2):e650-e659

Department of Gynecology and Obstetrics, Division of Reproductive Sciences & Women's Health Research, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Context: In vitro and in vivo evidence has supported the role of angiotensin II blockade in reducing leiomyoma cell proliferation and growth. However, no population-based study to date has investigated this potential association.

Objective: This work aims to determine whether prior angiotensin-converting enzyme inhibitor (ACEi) use is associated with a reduced odds of leiomyoma development.

Design: A nested case-control study was conducted.

Setting: The population was assembled from the Truven Health MarketScan Research Database, which includes private health insurance claims from January 1, 2012 to December 31, 2017.

Patients Or Other Participants: We included (n = 353 917) women age 18 to 65 with hypertension. Cases (n = 13 108) with a leiomyoma diagnosis were matched to controls (n = 340 808) with no such diagnosis at a 1:26 ratio by age and region of origin within the United States.

Intervention: Prior ACEi use was determined from outpatient drug claims.

Main Outcome Measure: Leiomyoma development was indicated by a first-time diagnosis code.

Results: Women on an ACEi experienced a 31.8% reduced odds of developing clinically recognized leiomyoma compared to nonusers (odds ratio [OR] 0.68; 95% CI, 0.65-0.72). This association was significant for each age group: 30 to 39 years (OR 0.86; 95% CI, 0.74-0.99), 40 to 49 years (OR 0.71; 95% CI, 0.66-0.76), 50 to 59 years (OR 0.63; 95% CI, 0.58-0.69), and 60 to 65 years (OR 0.58; 95% CI, 0.50-0.69). Of the ACEis, lisinopril (OR 0.67; 95% CI, 0.64-0.71), quinapril (OR 0.62; 95% CI, 0.41-0.92), and ramipril (OR 0.35; 95% CI, 0.23-0.50) demonstrated a significant association with reduced leiomyoma incidence.

Conclusions: ACEi use was associated with a reduced odds of developing clinically recognized leiomyoma in adult hypertensive women.
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http://dx.doi.org/10.1210/clinem/dgaa718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823233PMC
January 2021

SARS-CoV-2 outbreak investigation in a German meat processing plant.

EMBO Mol Med 2020 12 27;12(12):e13296. Epub 2020 Oct 27.

Viral Immune Modulation Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany.

We describe a multifactorial investigation of a SARS-CoV-2 outbreak in a large meat processing complex in Germany. Infection event timing, spatial, climate and ventilation conditions in the processing plant, sharing of living quarters and transport, and viral genome sequences were analyzed. Our results suggest that a single index case transmitted SARS-CoV-2 to co-workers over distances of more than 8 m, within a confined work area in which air is constantly recirculated and cooled. Viral genome sequencing shows that all cases share a set of mutations representing a novel sub-branch in the SARS-CoV-2 C20 clade. We identified the same set of mutations in samples collected in the time period between this initial infection cluster and a subsequent outbreak within the same factory, with the largest number of confirmed SARS-CoV-2 cases in a German meat processing facility reported so far. Our results indicate climate conditions, fresh air exchange rates, and airflow as factors that can promote efficient spread of SARS-CoV-2 via long distances and provide insights into possible requirements for pandemic mitigation strategies in industrial workplace settings.
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http://dx.doi.org/10.15252/emmm.202013296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7646008PMC
December 2020

Pushing beyond specifications: Evaluation of linearity and clinical performance of the cobas 6800/8800 SARS-CoV-2 RT-PCR assay for reliable quantification in blood and other materials outside recommendations.

J Clin Virol 2020 11 23;132:104650. Epub 2020 Sep 23.

University Medical Center Hamburg-Eppendorf (UKE), Institute of Medical Microbiology, Virology and Hygiene, Hamburg, Germany; German Center for Infection Research, Partner Site Hamburg-Borstel-Lübeck-Riems, Germany. Electronic address:

Background: The ongoing SARS-CoV-2 pandemic presents a unique challenge to diagnostic laboratories. There are preliminary studies correlating qRT-PCR results from different materials to clinical outcomes, yet, comparability is limited due to the plethora of different assays used for diagnostics. In this study we evaluate clinical performance and linear range for the SARS-CoV-2 IVD (cobas6800/8800 system, a fully automated sample-to-result platform) in different clinically relevant matrix materials outside official specifications.

Methods: Assay performance was assessed in human plasma, BAL/BL and transport medium following chemical inactivation. For analytical evaluation, respective matrix materials were spiked with SARS-CoV-2 RNA in ten-fold dilution series. The efficacy of chemical inactivation by guanidine hydrochloride solution was confirmed in cell culture infectivity experiments. For correlation, a total of 289 predetermined clinical samples including respiratory swabs, plasma and lower respiratory tract specimens were subjected to the SARS-CoV-2 IVD test and results were compared.

Results: The SARS-CoV-2 IVD showed excellent linearity over four to six log steps depending on matrix material. Chemical inactivation resulted in a reduction in plaque forming units of at least 3.5 log steps, while having no significant impact on assay performance. Inter-run consistency from three different testing sites demonstrated excellent comparability of RT-PCR results (maximum deviation was 1.53 CT). Clinical evaluation for respiratory swabs showed very good agreement with the comparator assay (Positive agreement 95.7 %, negative agreement 98.9 %).

Conclusion: The SARS-CoV-2 IVD test for the cobas6800/8800 systems offers excellent linear range and inter-run consistency for quantification of SARS-CoV-2 RNA in different matrices outside official specifications.
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http://dx.doi.org/10.1016/j.jcv.2020.104650DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510425PMC
November 2020

SARS Coronavirus-2 variant tracing within the first Coronavirus Disease 19 clusters in northern Germany.

Clin Microbiol Infect 2021 Jan 29;27(1):130.e5-130.e8. Epub 2020 Sep 29.

Institute for Medical Microbiology, Virology and Hygiene, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany. Electronic address:

Objectives: Investigation whether in depth characterization of virus variant patterns can be used for epidemiological analysis of the first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection clusters in Hamburg, Germany.

Methods: Metagenomic RNA-sequencing and amplicon-sequencing and subsequent variant calling in 25 respiratory samples from SARS-CoV-2 infected patients involved in the earliest infection clusters in Hamburg.

Results: Amplikon sequencing and cluster analyses of these SARS-CoV-2 sequences allowed the identification of the first infection cluster and five non-related infection clusters occurring at the beginning of the viral entry of SARS-CoV-2 in the Hamburg metropolitan region. Viral genomics together with epidemiological analyses revealed that the index patient acquired the infection in northern Italy and transmitted it to two out of 134 contacts. Single nucleotide polymorphisms clearly distinguished the virus variants of the index and other clusters and allowed us to track in which sequences worldwide these mutations were first described. Minor variant analyses identified the transmission of intra-host variants in the index cluster and household clusters.

Conclusions: SARS-CoV-2 variant tracing allows the identification of infection clusters and the follow up of infection chains occurring in the population. Furthermore, the follow up of minor viral variants in infection clusters can provide further resolution on transmission events indistinguishable at a consensus sequence level.
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http://dx.doi.org/10.1016/j.cmi.2020.09.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524521PMC
January 2021

High-resolution analysis of Merkel Cell Polyomavirus in Merkel Cell Carcinoma reveals distinct integration patterns and suggests NHEJ and MMBIR as underlying mechanisms.

PLoS Pathog 2020 08 24;16(8):e1008562. Epub 2020 Aug 24.

Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Merkel Cell Polyomavirus (MCPyV) is the etiological agent of the majority of Merkel Cell Carcinomas (MCC). MCPyV positive MCCs harbor integrated, defective viral genomes that constitutively express viral oncogenes. Which molecular mechanisms promote viral integration, if distinct integration patterns exist, and if integration occurs preferentially at loci with specific chromatin states is unknown. We here combined short and long-read (nanopore) next-generation sequencing and present the first high-resolution analysis of integration site structure in MCC cell lines as well as primary tumor material. We find two main types of integration site structure: Linear patterns with chromosomal breakpoints that map closely together, and complex integration loci that exhibit local amplification of genomic sequences flanking the viral DNA. Sequence analysis suggests that linear patterns are produced during viral replication by integration of defective/linear genomes into host DNA double strand breaks via non-homologous end joining, NHEJ. In contrast, our data strongly suggest that complex integration patterns are mediated by microhomology-mediated break-induced replication, MMBIR. Furthermore, we show by ChIP-Seq and RNA-Seq analysis that MCPyV preferably integrates in open chromatin and provide evidence that viral oncogene expression is driven by the viral promoter region, rather than transcription from juxtaposed host promoters. Taken together, our data explain the characteristics of MCPyV integration and may also provide a model for integration of other oncogenic DNA viruses such as papillomaviruses.
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http://dx.doi.org/10.1371/journal.ppat.1008562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7470373PMC
August 2020

Biomechanical Analysis of Plate Fixation Compared With Various Screw Configurations for Use in the Latarjet Procedure.

Orthop J Sports Med 2020 Jul 14;8(7):2325967120931399. Epub 2020 Jul 14.

Rothman Orthopaedics, New York, New York, USA.

Background: The biomechanical properties of coracoid fixation with a miniplate during the Latarjet procedure have not been described.

Purpose: To determine the biomechanical properties of miniplate fixation for the Latarjet procedure compared with various screw fixation configurations.

Study Design: Controlled laboratory study.

Methods: A total of 8 groups (n = 5 specimens per group) were tested at a screw insertion angle of 0°: (1) 3.75-mm single screw, (2) 3.75-mm double screw, (3) 3.75-mm double screw with washers, (4) 3.75-mm double screw with a miniplate, (5) 4.00-mm single screw, (6) 4.00-mm double screw, (7) 4.00-mm double screw with washers, and (8) 4.00-mm double screw with a miniplate. In addition, similar to groups 1 to 3 and 5 to 7, there were 30 additional specimens (n = 5 per group) tested at a screw insertion angle of 15° (groups 9-14). To maintain specimen uniformity, rigid polyurethane foam blocks were used. Testing parameters included a preload of 214 N for 10 seconds, cyclical loading from 184 to 736 N at 1 Hz for 100 cycles, and failure loading at a rate of 15 mm/min until 10 mm of displacement or specimen failure occurred.

Results: All single-screw constructs and 77% of 15° screw constructs failed before the completion of cyclical loading. Across all groups, group 8 (4.00-mm double screw with miniplate) demonstrated the highest maximum failure load ( < .001). There were no differences in failure loads among specimens with single-screw fixation (groups 1, 5, 9, and 12; > .05). All specimens in groups 9, 10, 11, 12, 13, and 14 (insertion angle of 15°) had significantly lower maximum failure loads compared with specimens in groups 2, 3, 4, 6, 7, and 8 (insertion angle of 0°) ( < .001 for all).

Conclusion: These results indicate significantly superior failure loads with the miniplate compared with all other constructs. Across all fixation techniques and screw sizes, constructs with screws inserted at 0° performed better than constructs with screws inserted at 15°.

Clinical Relevance: The use of a miniplate for coracoid fixation during the Latarjet procedure may provide a more durable construct for the high-demand contact athlete.
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http://dx.doi.org/10.1177/2325967120931399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7361494PMC
July 2020

Major central nervous system complications after allogeneic stem cell transplantation: A large retrospective study on 888 consecutive adult patients.

Eur J Haematol 2020 Dec 27;105(6):722-730. Epub 2020 Sep 27.

Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Objectives: Major complications affecting the central nervous system (CNS) present a challenge after allogeneic stem cell transplantation (allo-SCT).

Methods: Incidence, risk factors, and outcome were retrospectively analyzed in 888 patients in a monocentric study.

Results: Cumulative incidence (CI) of major CNS complications at 1 year was 14.8% (95%CI 12.3%-17.2%). Median follow-up is 11 months. CNS complications were documented in 132 patients: in 36 cases, classified metabolic; 26, drug-related neurotoxicity (14 attributed to cyclosporine A, 4 to antilymphocyte globulin); 11, cerebrovascular (ischemic n = 8, bleeding n = 3); 9, infections; 9, psychiatric; and 9, malignant. The cause of CNS symptoms remained unclear for 37 patients (28%). Multivariate analysis demonstrated an association of CNS complication with patient age (P < .001). The estimated OS of patients with any CNS complication was significantly lower than in patients without neurological complications (P < .001), and the CI of non-relapse mortality (NRM) was higher for patients with CNS complication (P < .001). A significant negative impact on survival can only be demonstrated for metabolic CNS complications and CNS infections (NRM, P < .0001 and P = .0003, respectively), and relapse (P < .0001).

Conclusion: CNS complications after allo-SCT are frequent events with a major contribution to morbidity and mortality. In particular, the situations of unclear neurological complications need to be clarified by intensive research.
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http://dx.doi.org/10.1111/ejh.13489DOI Listing
December 2020

Nutrition and physical activity recommendations from the United States and European cardiovascular guidelines: a comparative review.

Curr Opin Cardiol 2020 09;35(5):508-516

The Ciccarone Center for the Prevention of Cardiovascular Disease, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Purpose Of Review: A healthy lifestyle throughout one's lifespan is the core foundation for both primary and secondary prevention of cardiovascular disease (CVD). Risk-based decisions for pharmacological therapy is added on-top of lifestyle management. Thus, understanding lifestyle-based recommendations is central to CVD prevention.

Recent Findings: In 2018 and 2019, the American Heart Association (AHA) and American College of Cardiology (ACC) published new guidelines for lipid management and primary prevention of cardiovascular disease (CVD), respectively. The European Society of Cardiology (ESC) and European Atherosclerosis Society (EAS) published new guidelines on lipids and diabetes management in 2019. These guidelines provide recommendations on diet and lifestyle for reducing cardiovascular risk. Both encourage heart-healthy diets consistent with Mediterranean, DASH, or healthy vegetarian patterns. Both provide guidance for recommended physical activity levels but acknowledge any physical activity, even less than recommended, is better than inactivity. Although both ACC/AHA and ESC/EAS guidelines have similar approaches to achieve the same goal of CVD prevention, there were some differences between them.

Summary: In this review, we discussed similarities and differences between the American and European guidelines to familiarize clinicians with both sets of lifestyle recommendations in an effort to provide best practices in individualized patient-care for CVD prevention.
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http://dx.doi.org/10.1097/HCO.0000000000000763DOI Listing
September 2020

Alpha-1 adrenergic receptor antagonists for preventing acute respiratory distress syndrome and death from cytokine storm syndrome.

ArXiv 2020 Apr 21. Epub 2020 Apr 21.

In severe viral pneumonias, including Coronavirus disease 2019 (COVID-19), the viral replication phase is often followed by a hyperinflammatory reaction ('cytokine storm syndrome') that leads to acute respiratory distress syndrome and death, despite maximal supportive care. Preventing hyperinflammation is key to avoiding these outcomes. We previously demonstrated that alpha-1 adrenergic receptor antagonists ($\alpha$-blockers) can prevent cytokine storm syndrome and death in mice. Here, we conduct a retrospective analysis of patients with acute respiratory distress or pneumonia (n = 13,125 and n = 108,956, respectively) from all causes; patients who were incidentally taking $\alpha$-blockers had a reduced risk of requiring ventilation (by 35% and 16%, respectively), and a reduced risk of being ventilated and dying (by 56% and 20%, respectively), compared to non-users. Beta-adrenergic receptor antagonists had no significant effects. These results highlight the urgent need for prospective trials testing whether prophylactic $\alpha$-blockers improve outcomes in diseases with a prominent hyperinflammatory component such as COVID-19.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7280904PMC
April 2020

PVAmpliconFinder: a workflow for the identification of human papillomaviruses from high-throughput amplicon sequencing.

BMC Bioinformatics 2020 Jun 8;21(1):233. Epub 2020 Jun 8.

International Agency for Research on Cancer, Lyon, France.

Background: The detection of known human papillomaviruses (PVs) from targeted wet-lab approaches has traditionally used PCR-based methods coupled with Sanger sequencing. With the introduction of next-generation sequencing (NGS), these approaches can be revisited to integrate the sequencing power of NGS. Although computational tools have been developed for metagenomic approaches to search for known or novel viruses in NGS data, no appropriate tool is available for the classification and identification of novel viral sequences from data produced by amplicon-based methods.

Results: We have developed PVAmpliconFinder, a data analysis workflow designed to rapidly identify and classify known and potentially new Papillomaviridae sequences from NGS amplicon sequencing with degenerate PV primers. Here, we describe the features of PVAmpliconFinder and its implementation using biological data obtained from amplicon sequencing of human skin swab specimens and oral rinses from healthy individuals.

Conclusions: PVAmpliconFinder identified putative new HPV sequences, including one that was validated by wet-lab experiments. PVAmpliconFinder can be easily modified and applied to other viral families. PVAmpliconFinder addresses a gap by providing a solution for the analysis of NGS amplicon sequencing, increasingly used in clinical research. The PVAmpliconFinder workflow, along with its source code, is freely available on the GitHub platform: https://github.com/IARCbioinfo/PVAmpliconFinder.
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http://dx.doi.org/10.1186/s12859-020-03573-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7282039PMC
June 2020

Complete Genome Sequence of a SARS-CoV-2 Strain Isolated in Northern Germany.

Microbiol Resour Announc 2020 Jun 4;9(23). Epub 2020 Jun 4.

Institute for Medical Microbiology, Virology, and Hygiene, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Here, we describe the complete genome sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain isolated from an oropharyngeal swab sample from a female patient with COVID-19 who was infected in Hamburg, northern Germany.
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http://dx.doi.org/10.1128/MRA.00520-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272567PMC
June 2020

Clinical evaluation of a SARS-CoV-2 RT-PCR assay on a fully automated system for rapid on-demand testing in the hospital setting.

J Clin Virol 2020 07 28;128:104390. Epub 2020 Apr 28.

University Medical Center Hamburg-Eppendorf (UKE), Institute of Medical Microbiology, Virology and Hygiene, Hamburg, Germany.

Background: The ongoing SARS-CoV-2 pandemic presents a unique challenge for diagnostic laboratories around the world. Automation of workflows in molecular diagnostics is instrumental for coping with the large number of tests ordered by clinicians, as well as providing fast-tracked rapid testing for highly urgent cases. In this study we evaluated a SARS-CoV-2 LDT for the NeuMoDx 96 system, a fully automated device performing extraction and real-time PCR.

Methods: A publicly available SARS-CoV-2 RT-PCR assay was adapted for the automated system. Analytical performance was evaluated using in-vitro transcribed RNA and clinical performance was compared to the cobas 6800-based reference assay within the lab.

Results: The Envelope (E) Gene-LDT displayed good analytical performance with an LoD of 95.55 cp/mL and no false positives during evaluation of cross-reactivity. A total of 176 patient samples were tested with both the E-Gene-LDT and the reference assay. Positive and negative agreement were 100 % and 99.2 % respectively. Invalid-rate was 6.3 %.

Conclusion: The E-Gene-LDT showed analytical and clinical performance comparable to the cobas6800-based reference assay. Due to its random-access workflow concept and rapid time-to-result of about 80 min, the system is very well suited for providing fast-tracked SARS-CoV-2 diagnostics for urgent clinical samples in the hospital setting.
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http://dx.doi.org/10.1016/j.jcv.2020.104390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7187839PMC
July 2020

Infection-induced epigenetic changes and their impact on the pathogenesis of diseases.

Authors:
Nicole Fischer

Semin Immunopathol 2020 04;42(2):127-130

Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

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http://dx.doi.org/10.1007/s00281-020-00793-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7174260PMC
April 2020

Acute Generalized Cutaneous Lupus Erythematosus Repeatedly Mistaken for Cellulitis.

Cureus 2020 Feb 11;12(2):e6946. Epub 2020 Feb 11.

Internal Medicine, Nova Southeastern University Dr. Kiran C. Patel College of Osteopathic Medicine, Ft. Lauderdale, USA.

Cutaneous lupus erythematosus (CLE) may occur in association with systemic lupus erythematosus (SLE) or independently of SLE. Among the various subtypes of CLE, acute cutaneous lupus erythematosus (ACLE) has the highest rate of occurrence in association with SLE rather than independently; thus, if a patient presents with ACLE, a workup for SLE should be performed if not already diagnosed. In this case, we present a 52-year-old female with a past medical history consistent with a diagnosis of SLE (including end-stage renal disease, antiphospholipid syndrome, and seizure disorder); however, the patient went undiagnosed for years. Thus, when she presented with an unusual presentation of ACLE, the diagnosis was initially overlooked.
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http://dx.doi.org/10.7759/cureus.6946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7067358PMC
February 2020

Merkel Cell Polyomavirus DNA Replication Induces Senescence in Human Dermal Fibroblasts in a Kap1/Trim28-Dependent Manner.

mBio 2020 03 10;11(2). Epub 2020 Mar 10.

Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Merkel cell polyomavirus (MCPyV) is the only polyomavirus known to be associated with tumorigenesis in humans. Similarly to other polyomaviruses, MCPyV expresses a arge umor antigen (LT-Ag) that, together with a mall umor antigen (sT-Ag), contributes to cellular transformation and that is of critical importance for the initiation of the viral DNA replication. Understanding the cellular protein network regulated by MCPyV early proteins will significantly contribute to our understanding of the natural MCPyV life cycle as well as of the mechanisms by which the virus contributes to cellular transformation. We here describe KRAB-associated protein 1 (Kap1), a chromatin remodeling factor involved in cotranscriptional regulation, as a novel protein interaction partner of MCPyV T antigens sT and LT. Kap1 knockout results in a significant increase in the level of viral DNA replication that is highly suggestive of Kap1 being an important host restriction factor during MCPyV infection. Differently from other DNA viruses, MCPyV gene expression is unaffected in the absence of Kap1 and Kap1 does not associate with the viral genome. Instead, we show that in primary normal human dermal fibroblast (nHDF) cells, MCPyV DNA replication, but not T antigen expression alone, induces ataxia telangiectasia mutated (ATM) kinase-dependent Kap1 S824 phosphorylation, a mechanism that typically facilitates repair of double-strand breaks in heterochromatin by arresting the cells in G We show that MCPyV-induced inhibition of cell proliferation is mainly conferred by residues within the origin binding domain and thereby by viral DNA replication. Our data suggest that phosphorylation of Kap1 and subsequent Kap1-dependent G arrest/senescence represent host defense mechanisms against MCPyV replication in nHDF cells. We here describe Kap1 as a restriction factor in MCPyV infection. We report a novel, indirect mechanism by which Kap1 affects MCPyV replication. In contrast with from other DNA viruses, Kap1 does not associate with the viral genome in MCPyV infection and has no impact on viral gene expression. In MCPyV-infected nHDF cells, Kap1 phosphorylation (pKap1 S824) accumulates because of genomic stress mainly induced by viral DNA replication. In contrast, ectopic expression of LT or LT MCPyV mutants, previously shown to be important for induction of genotoxic stress, does not result in a similar extent of pKap1 accumulation. We show that cells actively replicating MCPyV accumulate pKap1 (in a manner dependent on the presence of ATM) and display a senescence phenotype reflected by G arrest. These results are supported by transcriptome analyses showing that LT antigen, in a manner dependent on the presence of Kap1, induces expression of secreted factors, which is known as the senescence-associated secretory phenotype (SASP).
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http://dx.doi.org/10.1128/mBio.00142-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064754PMC
March 2020

The evolution of the heart-healthy diet for vascular health: A walk through time.

Vasc Med 2020 04 3;25(2):184-193. Epub 2020 Mar 3.

The Ciccarone Center for the Prevention of Cardiovascular Disease, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

The rate of cardiovascular disease (CVD) mortality reduction in the United States has plateaued recently, despite the development of novel preventive pharmacotherapies, increased access to care, and healthcare spending. This is largely due to American's poor dietary patterns and practices causing increasing trends in the prevalence of obesity and type 2 diabetes mellitus. For decades, dietary guidelines on 'healthy diets' to reduce CVD risk, grounded in epidemiological research, have been nationally distributed to Americans. In this review, we highlight landmark events in modern nutrition science and how these have framed past and current understandings of diet and health. We also follow the evolution of dietary recommendations for Americans throughout the years, with an emphasis on recommendations aimed to reduce risk for CVD and mortality. Secondly, we examine how the low-fat ideology came to dominate America in the last decades of the 20th century and subsequently contributed to an excess intake of refined carbohydrates which, in the context of an increasingly sedentary lifestyle, may have fueled the obesity epidemic. We then examine the current major evidence-based dietary patterns and specific dietary approaches to reduce CVD risk, reviewing the literature surrounding nutritional components of the heart-healthy diet and discussing the dietary patterns proven most effective for CVD prevention: the Dietary Approaches to Stop Hypertension (DASH) diet, the Mediterranean diet, and the healthy vegetarian diet. Finally, we discuss emerging dietary trends, considerations for nutrition counseling, and future directions within the important field of nutrition, with the ultimate goal of improving vascular health.
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http://dx.doi.org/10.1177/1358863X19901287DOI Listing
April 2020

The Ubiquitin-Specific Protease Usp7, a Novel Merkel Cell Polyomavirus Large T-Antigen Interaction Partner, Modulates Viral DNA Replication.

J Virol 2020 02 14;94(5). Epub 2020 Feb 14.

Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Merkel cell polyomavirus (MCPyV) is the major cause for Merkel cell carcinoma (MCC), a rare but highly aggressive skin cancer predominantly found in elderly and immunosuppressed patients. The early viral gene products large T-antigen (LT) and small T-antigen (sT) are important for efficient viral DNA replication, and both contribute to transformation processes. These functions are executed mainly through interactions with host factors. Here, we identify the cellular ubiquitin-specific processing protease 7 (Usp7) as a new interaction partner of the MCPyV LT. Using glutathione -transferase pulldown experiments, we show that MCPyV LT directly binds to Usp7 and that N- as well as C-terminal regions of LT bind to the TRAF (tumor necrosis factor receptor-associated) domain of Usp7. We demonstrate that endogenous Usp7 coprecipitates with MCPyV T-antigens and relocalizes to viral DNA replication centers in cells actively replicating MCPyV genomes. We show that Usp7 does not alter ubiquitination levels of the T-antigens; however, Usp7 binding increases the binding affinity of LT to the origin of replication, thereby negatively regulating viral DNA replication. Together, these data identify Usp7 as a restriction factor of MCPyV replication. In contrast to other DNA viruses, Usp7 does not affect MCPyV gene expression via its ubiquitination activity but influences MCPyV DNA replication solely via a novel mechanism that modulates binding of LT to viral DNA. MCPyV is the only human polyomavirus that is associated with cancer; the majority of Merkel cell cancers have a viral etiology. While much emphasis was placed on investigations to understand the transformation process by MCPyV oncoproteins and cellular factors, we have only limited knowledge of cellular factors participating in the MCPyV life cycle. Here, we describe Usp7, a cellular deubiquitination enzyme, as a new factor involved in MCPyV replication. Usp7 is known in the context of large DNA tumor viruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus, to restrict viral replication. Similar to EBV, where Usp7 binding to EBNA1 increases EBNA1 binding affinity to viral DNA, we find MCPyV LT binding to the origin of replication to be increased in the presence of Usp7, resulting in restriction of viral DNA replication. However, Usp7-induced restriction of MCPyV replication is independent of its enzymatic activity, thereby constituting a novel mechanism of Usp7-induced restriction of viral replication.
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http://dx.doi.org/10.1128/JVI.01638-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7022344PMC
February 2020

DAMIAN: an open source bioinformatics tool for fast, systematic and cohort based analysis of microorganisms in diagnostic samples.

Sci Rep 2019 11 14;9(1):16841. Epub 2019 Nov 14.

Heinrich-Pette-Institute (HPI), Leibniz Institute for Experimental Virology, Research Group Virus Genomics, Hamburg, Germany.

We describe DAMIAN, an open source bioinformatics tool designed for the identification of pathogenic microorganisms in diagnostic samples. By using authentic clinical samples and comparing our results to those from established analysis pipelines as well as conventional diagnostics, we demonstrate that DAMIAN rapidly identifies pathogens in different diagnostic entities, and accurately classifies viral agents down to the strain level. We furthermore show that DAMIAN is able to assemble full-length viral genomes even in samples co-infected with multiple virus strains, an ability which is of considerable advantage for the investigation of outbreak scenarios. While DAMIAN, similar to other pipelines, analyzes single samples to perform classification of sequences according to their likely taxonomic origin, it also includes a tool for cohort-based analysis. This tool uses cross-sample comparisons to identify sequence signatures that are frequently present in a sample group of interest (e.g., a disease-associated cohort), but occur less frequently in control cohorts. As this approach does not require homology searches in databases, it principally allows the identification of not only known, but also completely novel pathogens. Using samples from a meningitis outbreak, we demonstrate the feasibility of this approach in identifying enterovirus as the causative agent.
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http://dx.doi.org/10.1038/s41598-019-52881-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856179PMC
November 2019

Characterization of porcine endogenous retrovirus particles released by the CRISPR/Cas9 inactivated cell line PK15 clone 15.

Xenotransplantation 2020 03 31;27(2):e12563. Epub 2019 Oct 31.

Division of Medical Biotechnology, Paul-Ehrlich-Institut, Langen, Germany.

The infection of human transplant recipients by porcine endogenous retrovirus (PERV) is a safety issue for xenotransplantation (XTx). CRISPR/Cas9 technology has enabled the generation of pigs free of functional PERVs, and the susceptibility of these animals to reinfection by PERVs remains unclear. To assess virological safety, we characterized a cell line in which PERVs have been inactivated by CRISPR/Cas9 (PK15 clone 15) for its susceptibility to infectious PERV. First, basal expression of PERV pol, the porcine PERV-A receptor (POPAR), and reverse transcriptase (RT) activity of PERV were determined. PK15 clone 15 cells were inoculated with PERV and monitored post infection for virus expression and RT activity. Particles were visualized by electron microscopy. Our data show that PK15 clone 15 cells still produce viral proteins that assemble to produce impaired viral particles. These virions have an irregular morphology that diverges from that of mature wild type. The particles are no longer infectious when tested in a downstream infection assay using supernatants of PK15 clone 15 cells to infect susceptible swine testis-IOWA (ST-IOWA) cells. The expression of POPAR was quantified to exclude the possibility that lack of susceptibility to reinfection, for PERV-A, is caused by absence of viral host receptor(s). PK15 and PK15 clone 15 cells do, in fact, express POPAR equally. PERV RT inactivation mediated by CRISPR/Cas9 does not compromise virus assembly but affects virion structure and proviral integration. The constitutive virion production seems to maintain cellular resistance to superinfection and possibly indicates a protective side effect of this specific CRISPR/Cas9 mediated RT inactivation.
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http://dx.doi.org/10.1111/xen.12563DOI Listing
March 2020