NASCarD (Nanopore Adaptive Sampling with Carrier DNA): A Rapid, PCR-Free Method for SARS-CoV-2 Whole-Genome Sequencing in Clinical Samples.

Whole-genome sequencing (WGS) represents the main technology for SARS-CoV-2 lineage characterization in diagnostic laboratories worldwide. The rapid, near-full-length sequencing of the viral genome is commonly enabled by high-throughput sequencing of PCR amplicons derived from cDNA molecules. Here, we present a new approach called NASCarD (Nanopore Adaptive Sampling with Carrier DNA), which allows a low amount of nucleic acids to be sequenced while selectively enriching for sequences of interest, hence limiting the production of non-target sequences. Using COVID-19 positive samples available during the omicron wave, we demonstrate how the method may lead to >99% genome completeness of the SARS-CoV-2 genome sequences within 7 h of sequencing at a competitive cost. The new approach may have applications beyond SARS-CoV-2 sequencing for other DNA or RNA pathogens in clinical samples.

Population-Based Severe Acute Respiratory Syndrome Coronavirus 2 Whole-Genome Sequencing and Contact Tracing During the Coronavirus Disease 2019 Pandemic in Switzerland.

BACKGROUND: Testing and contact tracing (CT) can interrupt transmission chains of SARS-CoV-2. Whole-genome sequencing (WGS) can potentially strengthen these investigations and provide insights on transmission. METHODS: We included all laboratory-confirmed COVID-19 cases diagnosed between 4 June and 26 July 2021, in a Swiss canton. We defined CT clusters based on epidemiological links reported in the CT data and genomic clusters as sequences with no single-nucleotide polymorphism (SNP) differences between any 2 pairs of sequences being compared. We assessed the agreement between CT clusters and genomic clusters. RESULTS: Of 359 COVID-19 cases, 213 were sequenced. Overall, agreement between CT and genomic clusters was low (Cohen's κ = 0.13). Of 24 CT clusters with ≥2 sequenced samples, 9 (37.5%) were also linked based on genomic sequencing but in 4 of these, WGS found additional cases in other CT clusters. Household was most often reported source of infection (n = 101 [28.1%]) and home addresses coincided well with CT clusters: In 44 of 54 CT clusters containing ≥2 cases (81.5%), all cases in the cluster had the same reported home address. However, only a quarter of household transmission was confirmed by WGS (6 of 26 genomic clusters [23.1%]). A sensitivity analysis using ≤1-SNP differences to define genomic clusters resulted in similar results. CONCLUSIONS: WGS data supplemented epidemiological CT data, supported the detection of potential additional clusters missed by CT, and identified misclassified transmissions and sources of infection. Household transmission was overestimated by CT.

The specific features of the developing T cell compartment of the neonatal lung are a determinant of respiratory syncytial virus immunopathogenesis.

The human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants, possibly due to the properties of the immature neonatal pulmonary immune system. Using the newborn lamb, a classical model of human lung development and a translational model of RSV infection, we aimed to explore the role of cell-mediated immunity in RSV disease during early life. Remarkably, in healthy conditions, the developing T cell compartment of the neonatal lung showed major differences to that seen in the mature adult lung. The most striking observation being a high baseline frequency of bronchoalveolar IL-4-producing CD4+ and CD8+ T cells, which declined progressively over developmental age. RSV infection exacerbated this pro-type 2 environment in the bronchoalveolar space, rather than inducing a type 2 response per se. Moreover, regulatory T cell suppressive functions occurred very early to dampen this pro-type 2 environment, rather than shutting them down afterwards, while γδ T cells dropped and failed to produce IL-17. Importantly, RSV disease severity was related to the magnitude of those unconventional bronchoalveolar T cell responses. These findings provide novel insights in the mechanisms of RSV immunopathogenesis in early life, and constitute a major step for the understanding of RSV disease severity.

Basophil-derived IL-4 promotes epicutaneous antigen sensitization concomitant with the development of food allergy.

BACKGROUND: Exaggerated thymic stromal lymphopoietin (TSLP) production and infiltration of basophils are associated with the pathogenesis of atopic dermatitis (AD), a recognized risk factor for the development of food allergies. Although TSLP and basophils have been implicated in promotion of food-induced allergic disorders in response to epicutaneous sensitization, the mechanisms by which TSLP-elicited basophils guide the progression of allergic inflammation in the skin to distant mucosal sites, such as the gastrointestinal tract, are poorly understood. OBJECTIVE: We sought to test the role of basophil-intrinsic IL-4 production in TH2 sensitization to food antigens in the skin and effector food-induced allergic responses in the gut. METHODS: Mice were epicutaneously sensitized with ovalbumin on an AD-like skin lesion, followed by intragastric antigen challenge to induce IgE-mediated food allergy. The requirement for basophil-derived IL-4 production for TH2 polarization and the pathogenesis of IgE-mediated food allergy was assessed in vitro by using coculture experiments with naive T cells and in vivo by using Il4 3'UTR mice that selectively lack IL-4 production in basophils. RESULTS: Epicutaneous food antigen sensitization is associated with infiltration of IL-4-competent innate immune cells to the skin, with basophils and eosinophils representing the predominant populations. In contrast to basophils, absence of eosinophils did not alter disease outcome. Coculture of IL-4-competent basophils together with dendritic cells and naive T cells was sufficient to promote TH2 polarization in an IL-4-dependent manner in vitro, whereas absence of basophil-intrinsic IL-4 production in vivo was associated with reduced food-induced allergic responses. CONCLUSION: TSLP-elicited basophils promote epicutaneous sensitization to food antigens and subsequent IgE-mediated food allergy through IL-4. Strategies to target the TSLP-basophil-IL-4 axis in patients with AD might lead to innovative therapies that can prevent the progression of allergies to distant mucosal sites.