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.

Enterovirus D68 (EV-D68) was detected in 93 patients from five European countries between 1 January 2019 and 15 January 2020, a season with expected low circulation. Patients were primarily children (n = 67, median age: 4 years), 59 patients required hospitalisation and five had severe neurologic manifestations. Phylogenetic analysis revealed two clusters in the B3 subclade and subclade A2/D. This circulation of EV-D68 associated with neurological manifestations stresses the importance of surveillance and diagnostics beyond expected peak years.

To investigate the relationship between neutralization escape and persistent high-level BK polyomavirus replication after kidney transplant (KTx), VP1 sequences were determined by Sanger and next-generation sequencing in longitudinal samples from KTx recipients with persistent high-level viruria (non-controllers) compared to patients who suppressed viruria (controllers). The infectivity and neutralization resistance of representative VP1 mutants were investigated using pseudotype viruses. In all patients, the virus population was initially dominated by wild-type VP1 sequences, then non-synonymous VP1 mutations accumulated over time in non-controllers. BC-loop mutations resulted in reduced infectivity in 293TT cells and conferred neutralization escape from cognate serum in five out of six non-controller patients studied. When taken as a group, non-controller sera were not more susceptible to neutralization escape than controller sera, so serological profiling cannot predict subsequent control of virus replication. However, at an individual level, in three non-controller patients the VP1 variants that emerged exploited specific “holes” in the patient’s humoral response. Persistent high-level BK polyomavirus replication in KTx recipients is therefore associated with the accumulation of VP1 mutations that can confer resistance to neutralization, implying that future BKPyV therapies involving IVIG or monoclonal antibodies may be more effective when used as preventive or pre-emptive, rather than curative, strategies.

Following the extensive non-pharmaceutical interventions (NPIs) and behavioral changes in the wake of the SARS-CoV-2 pandemic, an interseasonal rise in respiratory syncytial virus (RSV) cases was observed in Germany in 2021. The aim of this study was to characterize the local molecular epidemiology of RSV infections in comparison to the three pre-pandemic seasons. Additionally, clinical data were retrieved from patient charts to determine the clinical significance of RSV infections. RSV detections peaked in calendar week 40 of 2021, 18 weeks earlier than the usual peak observed in the three pre-pandemic seasons. Sequence analysis revealed a close phylogenetic relatedness regardless of the season of origin. A significantly higher amount of pediatric cases (88.9% of all cases, p < 0.001) was observed for season 2021/2022. For the pediatric cases, significant differences were observed for an increased number of siblings in the household (p = 0.004), a lower rate of fever (p = 0.007), and a reduced amount of co-infections (p = 0.001). Although the mean age of the adult patients was significantly younger (47.1 vs. 64.7, p < 0.001), high rates of comorbidities, lower respiratory tract infections and intensive care unit admissions prevailed. The NPIs in the wake of the SARS-CoV-2 pandemic had a tremendous impact on the epidemiologic characteristics and seasonality of RSV and warrant further epidemiologic studies of this important pathogen.

Background: The hepatitis B and D virus (HBV/HDV) hepatocyte entry inhibitor bulevirtide (BLV) has been available in Europe since July 2020, after the registrational trial MYR202. Real-life data on the efficacy and safety of BLV are sparse. Methods: We have analysed the course of treatment with BLV (2 mg/day) plus tenofovir disoproxil fumarate (TDF) (245 mg/day) in patients with chronic hepatitis delta (CHD). Virologic (≥2 log reduction in HDV RNA or suppression of HDV RNA below the lower limit of detection) and biochemical (normalisation of serum ALT) treatment responses after 24 weeks were defined according to the MYR202 trial. Results: Seven patients were recruited (four with liver cirrhosis Child–Pugh A). After 24 weeks, a virologic response was observed in five of seven and a biochemical response was seen in three of six patients with elevated serum ALT at baseline. Extended treatment data > 48 weeks were available in three cases: two presented with continuous virologic and biochemical responses and in one individual an HDV-RNA breakthrough was observed. Adverse effects were not recorded. Conclusions: The first real-life data of the approved dosage of 2 mg of BLV in combination with TDF confirm the safety, tolerability, and efficacy of the registrational trial MYR202 for a treatment period of 24 weeks and beyond.