Retrospective Genotyping of Enteroviruses Using a Diagnostic Nanopore Sequencing Workflow.

Enteroviruses are among the most common viruses pathogenic to humans. They are associated with various forms of disease, ranging from mild respiratory illness to severe neurological diseases. In recent years, an increasing number of isolated cases of children developing meningitis or encephalitis as a result of enterovirus infection have been reported, as well as discrete enterovirus D68 outbreaks in North America in 2014 and 2016. We developed an assay to rapidly genotype enteroviruses by sequencing a region within the VP1 gene using nanopore Flongles. We retrospectively analyzed enterovirus-/rhinovirus-positive clinical samples from the Zurich, Switzerland area mainly collected during two seasons in 2019/2020 and 2021/2022. Respiratory, cerebrospinal fluid, and stool samples were analyzed. Whole-genome sequencing was performed on samples with ambiguous genotyping results and enterovirus D68-positive samples. Out of 255 isolates, a total of 95 different genotypes were found. A difference in the prevalence of enterovirus and rhinovirus infections was observed for both sample type and age group. In particular, children aged 0-4 years showed a higher frequency of enterovirus infections. Comparing the respiratory seasons, a higher prevalence was found, especially for enterovirus A and rhinovirus A after the SARS-CoV-2 pandemic. The enterovirus genotyping workflow provides a rapid diagnostic tool for individual analysis and continuous enterovirus surveillance.

Exploring viral aetiology in upper respiratory tract infections: insights from metagenomic next-generation sequencing in Swiss outpatients before and during the SARS-CoV-2 pandemic.

AIMS OF THE STUDY: Upper respiratory tract infections are among the most common reasons for primary care consultations. They are diagnosed predominantly based on clinical assessment. Here, we investigated the benefit of viral metagenomic next-generation sequencing (mNGS) in an outpatient setting. METHODS: This prospective cross-sectional study included immunocompetent patients with acute upper respiratory tract infections. General practitioners collected pharyngeal swabs and demographic and clinical data. Specimens were analysed using viral mNGS and conventional tests. RESULTS: Two hundred seventy-seven patients were recruited by 21 general practitioners between 10/2019 and 12/2020, of which 91% had a suspected viral aetiology. For 138 patients (49.8%), mNGS identified one or more respiratory viruses. The mNGS showed a high overall agreement with conventional routine diagnostic tests. Rhinoviruses were the most frequently detected respiratory viruses (20.2% of patients). Viral mNGS reflected the influenza wave in early 2020 and the SARS-CoV-2 pandemic outbreak in Switzerland in March 2020. Notably, rhinoviruses continued to circulate despite non-pharmaceutical hygiene measures. CONCLUSIONS: Viral mNGS allowed the initial diagnosis to be retrospectively re-evaluated. Assuming reduced turnaround times, mNGS has the potential to directly guide the treatment of upper respiratory tract infections. On an epidemiological level, our study highlights the utility of mNGS in respiratory infection surveillance, allowing early detection of epidemics and providing information crucial for prevention.

Rapid and sensitive single-sample viral metagenomics using Nanopore Flongle sequencing.

The ability of viral metagenomic Next-Generation Sequencing (mNGS) to unbiasedly detect nucleic acids in a clinical sample is a powerful tool for advanced diagnosis of viral infections. When clinical symptoms do not provide a clear differential diagnosis, extensive laboratory testing with virus-specific PCR and serology can be replaced by a single viral mNGS analysis. However, widespread diagnostic use of viral mNGS is thus far limited by long sample-to-result times, as most protocols rely on Illumina sequencing, which provides high and accurate sequencing output but is time-consuming and expensive. Here, we describe the development of an mNGS protocol based on the more cost-effective Nanopore Flongle sequencing with decreased turnaround time and lower, yet sufficient sequencing output to provide sensitive virus detection. Sample preparation (6 h) and sequencing (2 h) times are substantially reduced compared to Illumina mNGS and allow detection of DNA/RNA viruses at low input (up to 33-38 cycle threshold of specific qPCR). Although Flongles yield lower sequencing output, direct comparison with Illumina mNGS on diverse clinical samples showed similar results. Collectively, the novel Nanopore mNGS approach is specifically tailored for use in clinical diagnostics and provides a rapid and cost-effective mNGS strategy for individual testing of severe cases.

Identification of cis- and trans-acting elements regulating calretinin expression in mesothelioma cells.

Calretinin (CALB2) is a diagnostic marker for epithelioid mesothelioma. It is also a prognostic marker since patients with tumors expressing high calretinin levels have better overall survival. Silencing of calretinin decreases viability of epithelioid mesothelioma cells. Our aim was to elucidate mechanisms regulating calretinin expression in mesothelioma. Analysis of calretinin transcript and protein suggested a control at the mRNA level. Treatment with 5-aza-2'-deoxycytidine and analysis of TCGA data indicated that promoter methylation is not likely to be involved. Therefore, we investigated CALB2 promoter by analyzing ~1kb of genomic sequence surrounding the transcription start site (TSS) + 1 using promoter reporter assay. Deletion analysis of CALB2 proximal promoter showed that sequence spanning the -161/+80bp region sustained transcriptional activity. Site-directed analysis identified important cis-regulatory elements within this -161/+80bp CALB2 promoter. EMSA and ChIP assays confirmed binding of NRF-1 and E2F2 to the CALB2 promoter and siRNA knockdown of NRF-1 led to decreased expression of calretinin. Cell synchronization experiment showed that calretinin expression was cell cycle regulated with a peak of expression at G1/S phase. This study provides the first insight in the regulation of CALB2 expression in mesothelioma cells.

Prevalence of BRCA-1 associated protein 1 germline mutation in sporadic malignant pleural mesothelioma cases.

OBJECTIVE: 23% of mesothelioma tumor specimens have a mutation in the BRCA1-associated protein 1 (BAP1) gene and germline BAP1 mutations predispose to malignant pleural mesothelioma (MPM). Our aim was to investigate germline BAP1 mutations in sporadic MPM patients. MATERIALS AND METHODS: Exonic DNA from peripheral blood leucocytes of 78 MPM patients was screened for germline BAP1 mutation. RESULTS: One out of 78 patients showed a germline synonymous mutation in exon 11. In all other patients wild-type sequence without any single-nucleotide polymorphisms was detected. CONCLUSIONS: Taking into account previous similar screenings, the prevalence of germline BAP1 mutations in sporadic MPM patients can be estimated around 1-2%, suggesting a minor role of germline BAP1 mutation in the pathogenesis of sporadic MPM.

GAS5 long non-coding RNA in malignant pleural mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is an aggressive cancer with short overall survival. Long non-coding RNAs (lncRNA) are a class of RNAs more than 200 nucleotides long that do not code for protein and are part of the 90% of the human genome that is transcribed. Earlier experimental studies in mice showed GAS5 (growth arrest specific transcript 5) gene deletion in asbestos driven mesothelioma. GAS5 encodes for a lncRNA whose function is not well known, but it has been shown to act as glucocorticoid receptor decoy and microRNA "sponge". Our aim was to investigate the possible role of the GAS5 in the growth of MPM. METHODS: Primary MPM cultures grown in serum-free condition in 3% oxygen or MPM cell lines grown in serum-containing medium were used to investigate the modulation of GAS5 by growth arrest after inhibition of Hedgehog or PI3K/mTOR signalling. Cell cycle length was determined by EdU incorporation assay in doxycycline inducible short hairpinGAS5 clones generated from ZL55SPT cells. Gene expression was quantified by quantitative PCR. To investigate the GAS5 promoter, a 0.77 kb sequence was inserted into a pGL3 reporter vector and luciferase activity was determined after transfection into MPM cells. Localization of GAS5 lncRNA was identified by in situ hybridization. To characterize cells expressing GAS5, expression of podoplanin and Ki-67 was assessed by immunohistochemistry. RESULTS: GAS5 expression was lower in MPM cell lines compared to normal mesothelial cells. GAS5 was upregulated upon growth arrest induced by inhibition of Hedgehog and PI3K/mTOR signalling in in vitro MPM models. The increase in GAS5 lncRNA was accompanied by increased promoter activity. Silencing of GAS5 increased the expression of glucocorticoid responsive genes glucocorticoid inducible leucine-zipper and serum/glucocorticoid-regulated kinase-1 and shortened the length of the cell cycle. Drug induced growth arrest was associated with GAS5 accumulation in the nuclei. GAS5 was abundant in tumoral quiescent cells and it was correlated to podoplanin expression. CONCLUSIONS: The observations that GAS5 levels modify cell proliferation in vitro, and that GAS5 expression in MPM tissue is associated with cell quiescence and podoplanin expression support a role of GAS5 in MPM biology.