Genomic studies on the impact of host/virus interaction in EBV infection using massively parallel high throughput sequencing

Epstein-Barr virus is one of the most common viral infections in humans and, once acquired, persists within its host throughout their life. EBV therefore represents an ex- tremely successful virus, having evolved complex strategies to evade the host’s innate and adaptive immune response during both initial and persistent stages of infection. While infection is mostly harmless in the majority of cases, EBV has the ability to be oncogenic in some individuals, and is associated with a wide range of malignancies as well as non-cancerous diseases. To generate new and useful insights into the evolution of EBV interactions with its host, a hybridization-based target enrichment methodology was optimised to enable whole genome sequencing of EBV directly from clinical samples. This allowed the gen- eration of whole genome sequences of EBV directly from blood for the first time. This methodology was subsequently applied to a number of distinct EBV sample col- lections and the resulting data used to investigate the intra- and inter-host variation in various clinical settings, such as infectious mononucleosis and immunosuppression with chronic EBV infection. Additionally, the number of available whole genomes from East Asia is expanded by eleven (unique) novel genomes from primary infection from a NPC- non-endemic area. These sequences were used for a comparative analysis between NPC- and non-NPC-derived EBV genomes and a number of sites were determined differenti- ating these two groups. Finally, comparative genomic analyses of world-wide EBV strain diversity were per- formed using genome sequences generated here in conjunction with a large number of publicly available EBV genome sequences. The comprehensive data sets generated, which included measures of diversity, selection, and linkage, were used to identify poten- tial targets of T cell immunity. In addition, the population structure of EBV was analysed to better understand the forces that have shaped the evolution of EBV

External quality assessment of SARS-CoV-2-sequencing: An ESGMD-SSM pilot trial across 15 European laboratories

Objective: This first pilot on external quality assessment (EQA) of SARS-CoV-2 whole genome sequencing, initiated by the ESCMID Study Group for Genomic and Molecular Diagnostics (ESGMD) and Swiss Society for Microbiology (SSM), aims to build a framework between laboratories in order to improve pathogen surveillance sequencing.Methods: Ten samples with varying viral loads were sent out to 15 clinical laboratories who had free choice of sequencing methods and bioinformatic analyses. The key aspects on which the individual centres were compared on were identification of 1) SNPs and indels, 2) Pango lineages, and 3) clusters between samples.Results: The participating laboratories used a wide array of methods and analysis pipelines. Most were able to generate whole genomes for all samples. Genomes were sequenced to varying depth (up to 100-fold difference across centres). There was a very good consensus regarding the majority of reporting criteria, but there were a few discrepancies in lineage and cluster assignment. Additionally, there were inconsistencies in variant calling. The main reasons for discrepancies were missing data, bioinformatic choices, and interpretation of data.Conclusions: The pilot EQA was an overall success. It was able to show the high quality of participating labs and provide valuable feedback in cases where problems occurred, thereby improving the sequencing setup of laboratories. A larger follow-up EQA should, however, improve on defining the variables and format of the report. Additionally, contamination and/or minority variants should be a further aspect of assessment.</p

Evaluation of the Dermal Toxicity of InZnP Quantum Dots Before and After Accelerated Weathering: Toward a Safer-By-Design Strategy

International audienceQuantum dots (QDs) are colloidal fluorescent semiconductor nanocrystals with exceptional optical properties. Their widespread use, particularly in light-emitting diodes (LEDs), displays, and photovoltaics, is questioning their potential toxicity. The most widely used QDs are CdSe and CdTe QDs, but due to the toxicity of cadmium (Cd), their use in electrical and electronic equipment is now restricted in the European Union through the Restriction of hazardous substances in electrical and electronic equipment (RoHS) directive. This has prompted the development of safer alternatives to Cd-based QDs; among them, InP QDs are the most promising ones. We recently developed RoHS-compliant QDs with an alloyed core composed of InZnP coated with a Zn(Se,S) gradient shell, which was further coated with an additional ZnS shell to protect the QDs from oxidative surface degradation. In this study, the toxicity of single-shelled InZnP/Zn(Se,S) core/gradient shell and of double-shelled InZnP/Zn(Se,S)/ZnS core/shell/shell QDs was evaluated both in their pristine form and after aging in a climatic chamber, mimicking a realistic environmental weathering. We show that both pristine and aged QDs, whatever their composition, accumulate in the cytoplasm of human primary keratinocytes where they form agglomerates at the vicinity of the nucleus. Pristine QDs do not show overt toxicity to cells, while aged QDs show cytotoxicity and genotoxicity and significantly modulate the mRNA expression of proteins involved in zinc homeostasis, cell redox response, and inflammation. While the three aged QDs show similar toxicity, the toxicity of pristine gradient-shell QD is higher than that of pristine double-shell QD, confirming that adding a second shell is a promising safer-by-design strategy. Taken together, these results suggest that end-of-life degradation products from InP-based QDs are detrimental to skin cells in case of accidental exposure and that the mechanisms driving this effect are oxidative stress, inflammation, and disturbance of cell metal homeostasis, particularly Zn homeostasis. Further efforts to promote safer-by-design formulations of QDs, for instance by reducing the In and Zn content and/or implementing a more robust outer shell, are therefore warranted

External quality assessment of SARS-CoV-2-sequencing: An ESGMD-SSM pilot trial across 15 European laboratories

OBJECTIVE This first pilot on external quality assessment (EQA) of SARS-CoV-2 whole genome sequencing, initiated by the ESCMID Study Group for Genomic and Molecular Diagnostics (ESGMD) and Swiss Society for Microbiology (SSM), aims to build a framework between laboratories in order to improve pathogen surveillance sequencing. METHODS Ten samples with varying viral loads were sent out to 15 clinical laboratories who had free choice of sequencing methods and bioinformatic analyses. The key aspects on which the individual centres were compared on were identification of 1) SNPs and indels, 2) Pango lineages, and 3) clusters between samples. RESULTS The participating laboratories used a wide array of methods and analysis pipelines. Most were able to generate whole genomes for all samples. Genomes were sequenced to varying depth (up to 100-fold difference across centres). There was a very good consensus regarding the majority of reporting criteria, but there were a few discrepancies in lineage and cluster assignment. Additionally, there were inconsistencies in variant calling. The main reasons for discrepancies were missing data, bioinformatic choices, and interpretation of data. CONCLUSIONS The pilot EQA was an overall success. It was able to show the high quality of participating labs and provide valuable feedback in cases where problems occurred, thereby improving the sequencing setup of laboratories. A larger follow-up EQA should, however, improve on defining the variables and format of the report. Additionally, contamination and/or minority variants should be a further aspect of assessment

Physico-Chemical Transformation and Toxicity of Multi-Shell InP Quantum Dots under Simulated Sunlight Irradiation, in an Environmentally Realistic Scenario

International audienceQuantum dots (QDs) are widely used in optoelectronics, lighting, and photovoltaics leadingto their potential release into the environment. The most promising alternative to the highly toxiccadmium selenide (CdSe) QDs are indium phosphide (InP) QDs, which show reduced toxicity andcomparable optical and electronic properties. QD degradation leads to the release of toxic metal ionsinto the environment. Coating the QD core with robust shell(s) composed of another semi-conductormaterial enhances their properties and protects the QD from degradation. We recently developeddouble-shelled InP QDs, which proved to be less toxic than single-shell QDs. In the present study,we confirm their reduced cytotoxicity, with an LC50 at 77 nM for pristine gradient shell QDs and>100 nM for pristine thin and thick shell QDs. We also confirm that these three QDs, when exposedto simulated sunlight, show greater cytotoxicity compared to pristine ones, with LC50 ranging from15 to 23 nM. Using a combination of spectroscopic and microscopic techniques, we characterize thedegradation kinetics and transformation products of single- and double-shell QDs, when exposedto solar light at high temperature, simulating environmental conditions. Non-toxic pristine QDsdegrade to form toxic In–phosphate, In–carboxylate, Zn–phosphate, and oxidized Se, all of whichprecipitate as heterogeneous deposits. Comparison of their degradation kinetics highlights that theQDs bearing the thickest ZnS outer shell are, as expected, the most resistant to photodegradationamong the three tested QDs, as gradient shell, thin shell, and thick shell QDs lose their opticalproperties in less than 15 min, 60 min, and more than 90 min, respectively. They exhibit the highestphotoluminescence efficiency, i.e., the best functionality, with a photoluminescence quantum yield inaqueous solution of 24%, as compared to 18% for the gradient shell and thin shell QDs. Therefore,they can be considered as safer-by-design QDs

Influence of the Core/Shell Structure of Indium Phosphide Based Quantum Dots on Their Photostability and Cytotoxicity

International audienceWith the goal to improve their photostability, InP-based QDs are passivated with three types of inorganic shells, namely (i) a gradient ZnSexS1-x shell, (ii) an additional ZnS shell on top of the gradient shell with two different thicknesses (core/shell/shell, CSS), (iii) an alumina coating on top of ZnS. All three systems have photoluminescence quantum yields (PLQY) > 50% and similar PL decay times (64-67 ns). To assess their photostability they are incorporated into a transparent poly (methyl methacrylate) (PMMA) matrix and exposed to continuous irradiation with simulated sunlight in a climate chamber. The alumina coated core/shell system exhibits the highest stability in terms of PLQY retention as well as the lowest shift of the PL maximum and lowest increase of the PL linewidth, followed by the CSS QDs and finally the gradient shell system. By means of XPS studies we identify the degradation of the ZnS outer layer and concomitant oxidation of the emissive InZnP core as the main origins of degradation in the gradient structure. These modifications do not occur in the case of the alumina-capped sample, which exhibits excellent chemical stability. The gradient shell and CSS systems could be transferred to the aqueous phase using surface ligand exchange with penicillamine. Cytotoxicity studies on human primary keratinocytes revealed that exposure for 24 h to 6.25-100 nM of QDs did not affect cell viability. However, a trend toward reduced cell proliferation is observed for higher concentrations of gradient shell and CSS QDs with a thin ZnS shell, while CSS QDs with a thicker ZnS shell do not exhibit any impact

PCR performance in the SARS-CoV-2 Omicron variant of concern?

Summary The new SARS-CoV-2 Omicron variant (B.1.1.529) has been recently declared a Variant of Concern due to a series of important mutations in the viral spike protein and especially in the receptor-binding domain. While investigations into the spread of this new variant are ongoing, the first cases have been detected in Switzerland. Important questions have been raised: (1) Will the PCR assays commonly used to detect SARS-CoV-2 still work for the Omicron variant? (2) Can specific PCR features, e.g. S-gene dropout, be used to identify potential Omicron samples? In this minireview we provide current knowledge on the Omicron variant and guidance on its PCR validation

EBV deletions as biomarkers of response to treatment of Chronic Active Epstein Barr Virus

Chronic active Epstein Barr Virus (CAEBV) is a rare condition occurring in previously healthy individuals associated with persistent EBV viraemia, fever, lymphadenopathy and hepatosplenomegaly. Viral deletions have been found in CAEBV and other lymphomas. However, it is unclear how stable these deletions are, whether they are present in different sites and how they evolve overtime. We sequenced fourteen longitudinal blood samples from three European CAEBV patients and compared with CAEBV saliva samples and other sequences from EBV-related conditions. We observed large EBV deletions in blood, but not saliva from CAEBV patients. Deletions were stable over time but were lost following successful treatment. Our results are consistent with the likelihood that certain deletions in the virus from CAEBV patients are associated with the evolution and persistence of haematological clones. We propose that the loss of deletions following successful treatment should be investigated as a potential biomarker to aid CAEBV management