Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples

Demultiplexed raw sequencing data files (fastq.gz

SARS-CoV-2 variants reveal features critical for replication in primary human cells

Since entering the human population, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2; the causative agent of Coronavirus Disease 2019 [COVID-19]) has spread worldwide, causing >100 million infections and >2 million deaths. While large-scale sequencing efforts have identified numerous genetic variants in SARS-CoV-2 during its circulation, it remains largely unclear whether many of these changes impact adaptation, replication, or transmission of the virus. Here, we characterized 14 different low-passage replication-competent human SARS-CoV-2 isolates representing all major European clades observed during the first pandemic wave in early 2020. By integrating viral sequencing data from patient material, virus stocks, and passaging experiments, together with kinetic virus replication data from nonhuman Vero-CCL81 cells and primary differentiated human bronchial epithelial cells (BEpCs), we observed several SARS-CoV-2 features that associate with distinct phenotypes. Notably, naturally occurring variants in Orf3a (Q57H) and nsp2 (T85I) were associated with poor replication in Vero-CCL81 cells but not in BEpCs, while SARS-CoV-2 isolates expressing the Spike D614G variant generally exhibited enhanced replication abilities in BEpCs. Strikingly, low-passage Vero-derived stock preparation of 3 SARS-CoV-2 isolates selected for substitutions at positions 5/6 of E and were highly attenuated in BEpCs, revealing a key cell-specific function to this region. Rare isolate-specific deletions were also observed in the Spike furin cleavage site during Vero-CCL81 passage, but these were rapidly selected against in BEpCs, underscoring the importance of this site for SARS-CoV-2 replication in primary human cells. Overall, our study uncovers sequence features in SARS-CoV-2 variants that determine cell-specific replication and highlights the need to monitor SARS-CoV-2 stocks carefully when phenotyping newly emerging variants or potential variants of concern

Nosocomial COVID-19 Incidence and Secondary Attack Rates among Patients of Tertiary Care Center, Zurich, Switzerland

Of 1,118 patients with COVID-19 at a university hospital in Switzerland during October 2020-June 2021, we found 83 (7.4%) had probable or definite healthcare-associated COVID-19. After in-hospital exposure, we estimated secondary attack rate at 23.3%. Transmission was associated with longer contact times and with lower cycle threshold values among index patients. Keywords: COVID-19; SARS-COV-2; Switzerland; coronavirus disease; cross-infection; infection control; infectious disease transmission; nosocomial infections; risk factors; severe acute respiratory syndrome coronavirus 2; viruses; zoonoses

Two Years of Viral Metagenomics in a Tertiary Diagnostics Unit: Evaluation of the First 105 Cases

Metagenomic next-generation sequencing (mNGS) can capture the full spectrum of viral pathogens in a specimen and has the potential to become an all-in-one solution for virus diagnostics. To date, clinical application is still in an early phase and limitations remain. Here, we evaluated the impact of viral mNGS for cases analyzed over two years in a tertiary diagnostics unit. High throughput mNGS was performed upon request by the treating clinician in cases where the etiology of infection remained unknown or the initial differential diagnosis was very broad. The results were compared to conventional routine testing regarding outcome and workload. In total, 163 specimens from 105 patients were sequenced. The main sample types were cerebrospinal fluid (34%), blood (33%) and throat swabs (10%). In the majority of the cases, viral encephalitis/meningitis or respiratory infection was suspected. In parallel, conventional virus diagnostic tests were performed (mean 18.5 individually probed targets/patients). mNGS detected viruses in 34 cases (32%). While often confirmatory, in multiple cases, the identified viruses were not included in the selected routine diagnostic tests. Two years of mNGS in a tertiary diagnostics unit demonstrated the advantages of a single, untargeted approach for comprehensive, rapid and efficient virus diagnostics, confirming the utility of mNGS in complementing current routine tests

Antibodies from convalescent plasma promote SARS-CoV-2 clearance in individuals with and without endogenous antibody response

BACKGROUNDNeutralizing antibodies are considered a key correlate of protection by current SARS-CoV-2 vaccines. The manner in which human infections respond to therapeutic SARS-CoV-2 antibodies, including convalescent plasma therapy, remains to be fully elucidated. METHODSWe conducted a proof-of-principle study of convalescent plasma therapy based on a phase I trial in 30 hospitalized COVID-19 patients with a median interval between onset of symptoms and first transfusion of 9 days (IQR, 7-11.8 days). Comprehensive longitudinal monitoring of the virological, serological, and disease status of recipients allowed deciphering of parameters on which plasma therapy efficacy depends. RESULTSIn this trial, convalescent plasma therapy was safe as evidenced by the absence of transfusion-related adverse events and low mortality (3.3%). Treatment with highly neutralizing plasma was significantly associated with faster virus clearance, as demonstrated by Kaplan-Meier analysis (P = 0.034) and confirmed in a parametric survival model including viral load and comorbidity (adjusted hazard ratio, 3.0; 95% CI, 1.1-8.1; P = 0.026). The onset of endogenous neutralization affected viral clearance, but even after adjustment for their pretransfusion endogenous neutralization status, recipients benefitted from plasma therapy with high neutralizing antibodies (hazard ratio, 3.5; 95% CI, 1.1-11; P = 0.034). CONCLUSIONOur data demonstrate a clear impact of exogenous antibody therapy on the rapid clearance of viremia before and after onset of the endogenous neutralizing response, and point beyond antibody-based interventions to critical laboratory parameters for improved evaluation of current and future SARS-CoV-2 therapies. TRIAL REGISTRATIONClinicalTrials.gov NCT04869072. FUNDINGThis study was funded via an Innovation Pool project by the University Hospital Zurich; the Swiss Red Cross Glückskette Corona Funding; Pandemiefonds of the UZH Foundation; and the Clinical Research Priority Program "Comprehensive Genomic Pathogen Detection" of the University of Zurich

Swiss public health measures associated with reduced SARS-CoV-2 transmission using genome data

Genome sequences from evolving infectious pathogens allow quantification of case introductions and local transmission dynamics. We sequenced 11,357 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from Switzerland in 2020 - the sixth largest effort globally. Using a representative subset of these data, we estimated viral introductions to Switzerland and their persistence over the course of 2020. We contrasted these estimates with simple null models representing the absence of certain public health measures. We show that Switzerland's border closures de-coupled case introductions from incidence in neighboring countries. Under a simple model, we estimate an 86-98% reduction in introductions during Switzerland's strictest border closures. Furthermore, the Swiss 2020 partial lockdown roughly halved the time for sampled introductions to die out. Last, we quantified local transmission dynamics once introductions into Switzerland occurred, using a phylodynamic model. We found that transmission slowed 35-63% upon outbreak detection in summer 2020, but not in fall. This finding may indicate successful contact tracing over summer before overburdening in fall. The study highlights the added value of genome sequencing data for understanding transmission dynamics

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

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

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

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

Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region, January to June 2020

We show the distribution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three genomic nomenclature systems to all sequence data from the World Health Organization European Region available until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation, compare the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2

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