Background: Understanding how children mount effective antiviral immune responses is critical for preparedness against current and future emerging pathogens. Acute respiratory viral infections provide a tractable human model to dissect age-specific immunity, yet the integration of humoral, cellular, and mucosal responses in children remains incompletely defined. Insights into cross-reactive and durable immune mechanisms are essential to inform vaccination strategies and pandemic readiness. Methods: 182 participants (96 children, 86 adults) from 41 families with newly confirmed SARS-CoV-2 infection were enrolled in the prospective OMI-Kids study (DRKS00029155). Repeated PCR testing, symptom monitoring, saliva sampling, and blood collection 6–8 weeks post household infection enabled detailed immune profiling. Humoral and cellular responses, including neutralization against Hu-1, BA.2, and EG.5.1, as well as HLA-restricted CD8+ T cell responses, were analyzed across distinct immunity profiles. Cytokine autoantibodies were quantified using a multiplex bead-based IgG assay. Findings: Serum and salivary IgG and IgA levels correlated strongly, supporting saliva as a reliable, non-invasive proxy for immune monitoring in children. Antibodies elicited by Hu-1–based infection or vaccination showed partial cross-reactivity to BA.2 but failed to neutralize EG.5.1. Despite limited antibody breadth, T cell-mediated immunity was conserved across variants, with no major age-dependent differences in the magnitude, HLA breadth or functionality of virus-specific T cell responses. Conclusion: These findings highlight fundamental features of antiviral immunity in children, characterized by constrained antibody breadth but robust and conserved cellular responses in the context of SARS-CoV-2 evolution. Cross-reactive T cell immunity may represent a key mechanism of protection against severe disease despite ongoing viral evolution. This work establishes a framework for leveraging acute viral infections as a human model to inform immune protection, surveillance strategies, and vaccine design for future emerging pathogens.

OBJECTIVES:Respiratory syncytial virus (RSV) is a major cause of morbidity globally, with most cases managed in outpatient settings. However, data characterizing the disease burden of non-severe RSV infections remain limited. METHODS:The prospective, multicenter pediatric airway pathogen incidence (PAPI) study enrolled 1450 children aged ≤24 months presenting with lower respiratory tract infections (LRTI) in outpatient settings between October 2021 and April 2023. Clinical data, detected pathogens via multiplex polymerase chain reaction, and follow-up data were collected. RESULTS:Among enrolled patients 19.7% tested positive for RSV. RSV-positive patients (RSV+) exhibited distinct clinical characteristics, including significantly higher proportions of wheezing, crackles, and tachypnoea than RSV-negative patients. Notably, we identified a previously unreported association between RSV infection and atopic conditions, with RSV+ patients demonstrating higher proportions of atopic eczema (odds ratio 1.65 [95% CI: 1.05-2.61]). 96.5% of all RSV+ patients received pharmacotherapy, predominantly beta-sympathomimetics, inhaled saline, and antipyretics. The mean recovery time was 12 days. CONCLUSION:In the outpatient setting, RSV infections during the first 2 years of life present with distinct clinical features and increased morbidity compared with other respiratory pathogens. The association with atopic conditions warrants further investigation. These findings characterize the considerable RSV burden in primary care before the implementation of immunization measures. TRIAL REGISTRATION:DRKS00026155.

BACKGROUND:Chikungunya fever is a mosquito-borne disease characterised by severe arthralgia and is caused by chikungunya virus (CHIKV), an alphavirus. The aim of this study was to establish a WHO International Standard for CHIKV-specific neutralising antibodies-a potential correlate of protection. METHODS:Two candidate International Standards (code numbers 1502/19 and 1504/19) were prepared: 1502/19 using plasma from a CHIKV-convalescent individual and 1504/19 using plasma from blood donors from a CHIKV-endemic region. Potencies of the two candidate International Standards were evaluated alongside samples from recovered individuals and donors previously infected with CHIKV, in an international study (2019-20), using a range of virus-neutralisation assays and immunoassays. Statistical analysis was used to estimate mean and relative potencies of the candidate International Standards. Participants included vaccine developers, arbovirus reference laboratories, and diagnostics manufacturers. FINDINGS:26 laboratories from 12 countries reported detection of anti-CHIKV antibodies and associated potencies. All participants consistently detected both 1502/19 and 1504/19. Overall mean potencies were near-identical for the two candidate International Standards (1502/19, 3·44 log10 and 1504/19, 3·48 log10). Intra-assay variation was considerably lower than inter-assay variation for the candidate International Standards, which varied by more than 100-fold; this variability was seen for all other anti-CHIKV-positive samples. Assay variability was substantially reduced when antibody titres from the panel of samples were expressed relative to 1502/19 or 1504/19. An agreement was reached to establish 1502/19 as the first WHO International Standard for anti-CHIKV neutralising antibodies, with an assigned unitage of 1000 International Unitage/mL. INTERPRETATION:The use of the candidate International Standards resulted in harmonisation of anti-CHIKV neutralisation titres, allowing for better comparison of results of neutralisation assays for anti-CHIKV antibodies, either as a consequence of natural infection or produced in response to different vaccines, and will be essential in estimating antibody titres associated with protection against CHIKV. FUNDING:German Ministry of Health (Bundesministerium für Gesundheit).

Severe HSV-1 disease is treated with potent antiviral drugs, in particular aciclovir (ACV) and its derivatives. However, long-term drug exposure in immunocompromised patients can lead to the emergence of ACV-resistant HSV-1 strains and clinical treatment failure. To understand how phenotypic resistances develop on a genomic level, we analyzed the influence of ACV selection pressure on the viral genome of different HSV-1 virus strains in vitro. Growth kinetics and IC50 determination showed ACV resistance development within a single passage. Next, we performed ultra-deep, non-targeted full-genome Illumina sequencing of the parental and ACV-adapted HSV-1 strains. Interestingly, resistance-conferring mutations rapidly arose in the viral genes UL23 and UL30 and were already present in the parental ACV-naïve strains at extremely low variant frequencies. Based on these findings, we hypothesized that low- frequency mutations develop during continued viral replication. To test this hypothesis, a primary rescued recombinant K17 + strain was repeatedly passaged. Continued passaging indeed increased the proportion of a subset of minor variants and allowed resistance development after, but not before, 10 consecutive passages. In summary, we show that minor variants can facilitate adaptation of HSV-1 populations to selective pressures such as pharmacological inhibition of replication. These findings highlight that deep sequencing might allow early detection of resistance mutations potentially supporting antiviral drug stewardship.

The majority of SARS-CoV-2 genomes obtained during the pandemic were derived by amplifying overlapping windows of the genome (‘tiled amplicons’), reconstructing their sequences and fitting them together. This leads to systematic errors in genomes unless the software is both aware of the amplicon scheme and of the error modes of amplicon sequencing. Additionally, over time, amplicon schemes need to be updated as new mutations in the virus interfere with the primer binding sites at the end of amplicons. Thus, waves of variants swept the world during the pandemic and were followed by waves of systematic errors in the genomes, which had significant impacts on the inferred phylogenetic tree. Here we reconstruct the genomes from all public data as of June 2024 using an assembly tool called Viridian ( https://github.com/iqbal-lab-org/viridian ), developed to rigorously process amplicon sequence data. With these high-quality consensus sequences we provide a global phylogenetic tree of 4,471,579 samples, viewable at https://viridian.taxonium.org . We provide simulation and empirical validation of the methodology, and quantify the improvement in the phylogeny.

With respiratory syncytial virus vaccines recently approved for use among older adults, country-level respiratory syncytial virus (RSV) disease burden estimates are needed to inform local RSV immunisation strategy. We aimed to estimate country-level RSV hospitalisation burden in older adults in Europe. We compiled data on RSV hospitalisation burden in adults aged ≥ 60 years in Europe from published studies (systematic review: PROSPERO CRD42024516945), surveillance data, and unpublished data from international collaborators. We adjusted for diagnostic testing, clinical specimens, and case definitions through statistical modelling techniques and generated country-level hospitalisation rate estimates; for countries with no available data, we developed an ensemble model to predict RSV hospitalisation rates. We also estimated RSV in-hospital case fatality ratio (hCFR) for countries with available data. We included 14 studies (3 unpublished studies). The adjusted RSV-associated hospitalisation rates were overall 2.2 to 6.4 times higher than unadjusted estimates. Among 5 countries with available data, adjusted annual RSV hospitalisation rates ranged from 193/100,000 person-years in the Netherlands (95

A 48-year-old patient underwent lung transplantation because of severe COVID-19, which aggravated his underlying interstitial lung disease, despite the presence of detectable SARS-CoV-2. Subsequently, the graft is re-infected early in the post-procedural phase, leading to viral persistence for more than five months. By analyzing viral evolution and effector immune response within the transplanted organ, we observe three main findings. First, virus evolution differs in the transplanted organ compared to that in the upper respiratory tract and is affected by monoclonal SARS-CoV-2-specific antibodies and molnupiravir. Second, we show the potential clinical relevance of T cell HLA restriction that may facilitate viral clearance in the upper respiratory tract compared to the ongoing viral replication in the HLA mismatch organ. Third, close monitoring and modulation of immunosuppressive and antiviral therapy enables viral clearance in a lung transplantation setting despite incomplete SARS-CoV-2 clearance prior to transplantation.

BACKGROUND:Antiviral drug resistance in herpes simplex virus 1 and 2 (HSV-1 and 2) is a significant clinical challenge, particularly in immunocompromised patients. Drug susceptibility testing (DST) aids clinical management and can be conducted through genotypic (partial genome sequencing) or phenotypic (cell culture) methods. Both have inherent limitations: genotypic DST is limited by outdated datasets lacking information on new helicase-primase inhibitors and corresponding phenotypic data as well as sparse clinical correlations. Phenotypic DST is mainly hampered by a lack of standardization and timely results. OBJECTIVES:This study aims to compile an up-to-date and comprehensive HSV drug resistance dataset encompassing all reported drug resistance-associated mutations (DRMs), polymorphisms, and viral phenotypes. This study also aims to aggregate clinical conditions with available DST data. SOURCES:A PubMed search identified studies (January 2016-September 2024) on DRMs associated with resistance to aciclovir, penciclovir, brivudine, foscarnet, cidofovir, amenamevir, and pritelivir. Data from a previous HSV resistance dataset (pre-2016) were also included. CONTENT:In this review, we summarize novel mutations in the thymidine kinase, polymerase, and helicase-primase genes of HSV conferring resistance to antiviral drugs. Clinical information was available for 513 mutations. In 90% of these (461 cases), viral phenotype and clinical assessment were congruent. However, 10% of cases not responding to antiviral therapy showed phenotypically susceptible virus isolates. We present a framework for clinical and diagnostic management of cases with drug-resistant HSV infection. IMPLICATIONS:This dataset paves the way to harmonize reporting of DRMs for diagnostic labs and to accelerate genotypic DST interpretation through aggregated data. Ongoing large-scale data collection of genotypic, phenotypic, and clinical data is crucial for evidence-based management of HSV antiviral resistance and clinical guidelines.

OBJECTIVES:Since 2022, distinct Mpox virus (MPXV) clades have been spreading across different geographic regions, causing a challenging epidemiological situation. Whole genome sequencing (WGS) proved to be instrumental for patient management and global public health. We report a pilot interlaboratory comparison study for MPXV WGS. METHODS:We distributed noninfectious DNA samples, including the main MPXV clades I and II, to eight European laboratories. We included one cowpox (CPXV) sample as a specificity control. Participants were free to choose their WGS pipeline of choice to mimic a real-world scenario and were asked to report on the sequencing pipeline used, average genome coverage, and MPXV species, clade, and subclade assignments. RESULTS:Seven of the eight invited laboratories reported results back. All participants largely identified the MPXV clades and reported high-quality genomes with minimal variations, specifically for MPXV clade IIb 2022 outbreak strains. However, reconstructed genomes showed high variability for nonclade IIb MPXV strains. The CPXV sample was correctly identified by three laboratories. CONCLUSIONS:Although results for MPXV clade IIb 2022 outbreak strains are reassuring, the inclusion of MPXV clade I and IIa strains highlights pitfalls for targeted sequencing approaches and subsequent bioinformatic analyses. Our findings underscore the need for standardized external quality assessment studies.

BACKGROUND:Nosocomial pneumonia, encompassing hospital-acquired (HAP) and ventilator-associated pneumonia (VAP), remains a major cause of morbidity and mortality in hospitalized adults. In response to evolving pathogen profiles and emerging resistance patterns, this updated S3 guideline (AWMF Register No. 020-013) provides an evidence-based framework to enhance the diagnosis, risk stratification, and treatment of nosocomial pneumonia. METHODS:The guideline update was developed by a multidisciplinary panel representing key German professional societies. A systematic literature review was conducted with subsequent critical appraisal using the GRADE methodology. Structured consensus conferences and external reviews ensured that the recommendations were clinically relevant, methodologically sound, and aligned with current antimicrobial stewardship principles. RESULTS:For the management of nosocomial pneumonia patients should be divided in those with and without risk factors for multidrug-resistant pathogens and/or Pseudomonas aeruginosa. Bacterial multiplex-polymerase chain reaction (PCR) should not be used routinely. Bronchoscopic diagnosis is not considered superior to non-bronchoscopic sampling in terms of main outcomes. Combination antibiotic therapy is now reserved for patients in septic shock and high risk for multidrug-resistant pathogens, while select patients may be managed with monotherapy (e. g., meropenem). In clinically stabilized patients, antibiotic therapy should be de-escalated and focused, as well as duration shortened to 7-8 days. In critically ill patients, prolonged application of suitable beta-lactam antibiotics should be preferred. Patients on the intensive care unit (ICU) are at risk for invasive pulmonary aspergillosis (IPA). Diagnostics for Aspergillus should be performed with an antigen test from bronchial lavage fluid. CONCLUSION:This updated S3 guideline offers a comprehensive, multidisciplinary approach to the management of nosocomial pneumonia in adults. By integrating novel diagnostic modalities and refined therapeutic strategies, it aims to standardize care, improve patient outcomes, and enhance antimicrobial stewardship to curb the emergence of resistant pathogens.

BACKGROUND:The emergence of resistance-associated substitutions in RSV against novel monoclonal antibodies is a concern given widespread prophylactic use. AIM:To assess the prevalence of resistance-associated substitutions in the RSV F protein against nirsevimab, clesrovimab, and palivizumab in German infants before widespread implementation of nirsevimab. MATERIALS & METHODS:We sequenced the F protein of n = 1042 RSV samples from German infants from seasons 2021/2022 and 2022/2023 and screened for variants in binding sites for nirsevimab (Site Ø), clesrovimab (Site IV), and palivizumab (Site II). RESULTS:Prevalence of resistance-associated substitutions was low (< 1%) for all three monoclonal antibodies. CONCLUSION:Although the current risk of infections with escape-mutants appears to be low, our results underline the need for continued surveillance, as resistance-conferring mutations to new mAbs circulated and may be selected under selection pressure.