Evolution of the Cytomegalovirus RL11 gene family in Old World monkeys and Great Apes.

Cytomegalovirus (CMV) is a genus of herpesviruses, members of which share a long history of coevolution with their primate hosts including New World monkeys, Old World monkeys (OWMs), and Great Apes (GAs). These viruses are ubiquitous within their host populations and establish lifelong infection in most individuals. Although asymptomatic in healthy individuals, infection poses a significant risk to individuals with a weakened or underdeveloped immune system. The genome of human CMV is the largest among human-infecting viruses and comprises at least 15 separate gene families, which may have arisen by gene duplication. Within human CMV, the RL11 gene family is the largest. RL11 genes are nonessential in vitro but have immune evasion roles that are likely critical to persistence in vivo. These genes demonstrate an extreme level of inter-species and intra-strain sequence diversity, which makes it challenging to deduce the evolutionary relationships within this gene family. Understanding the evolutionary relationships of these genes, especially accurate ortholog identification, is essential for reconstructing ancestral genomes, deciphering gene repertoire and order, and enabling reliable functional analyses across the CMV species, thereby offering insights into evolutionary processes, genetic diversity, and the functional significance of genes. In this work, we combined in silico genome screening with sequence-based and structure-guided phylogenetic analysis to reconstruct the evolutionary history of the RL11 gene family. We confirmed that RL11 genes are unique to OWM and GA CMVs, showing that this gene family was formed by multiple early duplication events and later lineage-specific losses. We identified four main clades of RL11 genes and showed that their expansions were mainly lineage specific and happened independently in CMVs of GAs, African OWMs, and Asian OWMs. We also identified groups of orthologous genes across the CMV tree, showing that some human CMV-specific RL11 genes emerged before the divergence of human and chimpanzee CMVs but were subsequently lost in the latter. The extensive and dynamic species-specific evolution of this gene family suggests that their functions target elements of host immunity that have similarly coevolved during speciation.

The Fibrotic Phenotype of Human Precision-Cut Lung Slices Is Maintained after Cryopreservation.

Human precision-cut lung slices (hPCLS) prepared from fibrotic lungs recapitulate the pathophysiological hallmarks of fibrosis. These hallmark features can also be induced by treating non-fibrotic hPCLS with a fibrotic cocktail (FC). As a result, the fibrotic and fibrosis-induced hPCLS are rapidly emerging as preferred models for disease modeling and drug discovery. However, current hPCLS models are limited by tissue viability in culture, as they are usually only viable for one week after harvesting. Here, we demonstrate that the fibrotic hPCLS can be cryopreserved, stored for months, and then thawed on demand without loss of hPCLS viability or protein content for 14 days post-thawing. Cryopreservation also preserves the pro-fibrotic potential of non-fibrotic hPCLS. Specifically, when we treated the thawed non-fibrotic hPCLS with an FC, we observed significant pro-fibrotic cytokine secretion and elevated tissue stiffness. These pro-fibrotic changes were inhibited by the small-molecule tyrosine kinase inhibitor, Nintedanib. Taken together, our work indicates that a feasible solution to prolong the pre-clinical utility of fibrotic and fibrosis-induced hPCLS is cryopreservation. We anticipate that cryopreserved hPCLS will serve as an advantageous predictive model for the evaluation of pro-fibrotic pathways during acute and chronic toxicity testing.

Near infra-red luminescent osmium labelled gold nanoparticles for cellular imaging and singlet oxygen generation.

Osmium(II) complexes have attractive properties for potential theranostic agents given their anticancer activitiy, their redox potentials favourable for biological transformations within cancer cells and their luminescence in the near infrared (NIR) region. To achieve localised detection and delivery, gold nanoparticles (AuNP) provide an attractive scaffold to attach multiple luminescent agents on a single particle and provide a multimodal platform for detection and loaclaised delivery. We have developed 13 nm and 25 nm AuNP decorated with an osmium complex based on 1,10-phenantholine and surface active bipyridine ligands, OsPhenSS for live cell imaging and singlet oxygen generation, notated as OsPhenSS·AuNP13 and OsPhenSS·AuNP25. The AuNP designs not only allow versatile modalities for localisation of the probe but also water solubility for the osmium metal complex. The osmium decorated nanoparticles OsPhenSS·AuNP13 and OsPhenSS·AuNP25 display characteristic NIR luminescence from the osmium(II) 3MLCT at 785 nm in aqueous solutions with visible excitation. Upon incubation of the nanoparticles in lung cancer and breast carcinoma the luminescence signature of osmium and the gold reflectance reveal localisation in the cytoplasmic and perinuclear compartments. Excitation of the nanoparticles at 552 nm in the presence of a ROS indicator revealed a marked increase in the green fluorescence from the indicator, consistent with photo-induced ROS generation. The detection of singlet oxygen by time-resolved luminescence studies of the osmium and the nanoparticle probes further demonstrates the dual activity of the osmium-based nanoprobes for imaging and therapy. The introduction of gold nanoparticles for carrying osmium imaging probes allows a novel versatile strategy combining detection and localised therapies at the nanoscale.

A novel approach to exploring the dark genome and its application to mapping of the vertebrate virus fossil record.

BACKGROUND: Genomic regions that remain poorly understood, often referred to as the dark genome, contain a variety of functionally relevant and biologically informative features. These include endogenous viral elements (EVEs)-virus-derived sequences that can dramatically impact host biology and serve as a virus fossil record. In this study, we introduce a database-integrated genome screening (DIGS) approach to investigate the dark genome in silico, focusing on EVEs found within vertebrate genomes. RESULTS: Using DIGS on 874 vertebrate genomes, we uncover approximately 1.1 million EVE sequences, with over 99% originating from endogenous retroviruses or transposable elements that contain EVE DNA. We show that the remaining 6038 sequences represent over a thousand distinct horizontal gene transfer events across 10 virus families, including some that have not previously been reported as EVEs. We explore the genomic and phylogenetic characteristics of non-retroviral EVEs and determine their rates of acquisition during vertebrate evolution. Our study uncovers novel virus diversity, broadens knowledge of virus distribution among vertebrate hosts, and provides new insights into the ecology and evolution of vertebrate viruses. CONCLUSIONS: We comprehensively catalog and analyze EVEs within 874 vertebrate genomes, shedding light on the distribution, diversity, and long-term evolution of viruses and reveal their extensive impact on vertebrate genome evolution. Our results demonstrate the power of linking a relational database management system to a similarity search-based screening pipeline for in silico exploration of the dark genome.

Luminescence Lifetime-Based Sensing Platform Based on Cyclometalated Iridium(III) Complexes for the Detection of Perfluorooctanoic Acid in Aqueous Samples.

Luminescence lifetimes are an attractive analytical method for detection due to its high sensitivity and stability. Iridium probes exhibit luminescence with long excited-state lifetimes, which are sensitive to the local environment. Perfluorooctanoic acid (PFOA) is listed as a chemical of high concern regarding its toxicity and is classified as a "forever chemical". In addition to strict limits on the presence of PFOA in drinking water, environmental contamination from industrial effluent or chemical spills requires rapid, simple, accurate, and cost-effective analysis in order to aid containment. Herein, we report the fabrication and function of a novel and facile luminescence sensor for PFOA based on iridium modified on gold surfaces. These surfaces were modified with lipophilic iridium complexes bearing alkyl chains, namely, IrC6 and IrC12, and Zonyl-FSA surfactant. Upon addition of PFOA, the modified surfaces IrC6-FSA@Au and IrC12-FSA @Au show the largest change in the red luminescence signal with changes in the luminescence lifetime that allow monitoring of PFOA concentrations in aqueous solutions. The platform was tested for the measurement of PFOA in aqueous samples spiked with known concentrations of PFOA and demonstrated the capacity to determine PFOA at concentrations >100 µg/L (240 nM).

MODFLOW as a Configurable Multi-Model Hydrologic Simulator.

MODFLOW 6 is the latest in a line of six "core" versions of MODFLOW released by the U.S. Geological Survey. The MODFLOW 6 architecture supports incorporation of additional hydrologic processes, in addition to groundwater flow, and allows interaction between processes. The architecture supports multiple model instances and multiple types of models within a single simulation, a flexible approach to formulating and solving the equations that represent hydrologic processes, and recent advances in interoperability, which allow MODFLOW to be accessed and controlled by external programs. The present version of MODFLOW 6 consolidates popular capabilities available in MODFLOW variants, such as the unstructured grid support in MODFLOW-USG, the Newton-Raphson formulation in MODFLOW-NWT, and the support for partitioned stress boundaries in MODFLOW-CDSS. The flexible multi-model capability allows users to configure MODFLOW 6 simulations to represent the local-grid refinement (LGR) capabilities available in MODFLOW-LGR, the multi-species transport capabilities in MT3DMS, and the coupled variable-density capabilities available in SEAWAT. This paper provides a new, holistic and integrated overview of simulation capabilities made possible by the MODFLOW 6 architecture, and describes how ongoing and future development can take advantage of the program architecture to integrate new capabilities in a way that is minimally invasive and automatically compatible with the existing MODFLOW 6 code.

An Agricultural Package for MODFLOW 6 Using the Application Programming Interface.

An agricultural water use package has been developed for MODFLOW 6 using the MODFLOW Application Programming Interface (API). The MODFLOW API Agricultural Water Use Package (API-Ag) was based on the approach to simulate irrigation demand in the MODFLOW-NWT and GSFLOW Agricultural Water Use (AG) Package. The API-Ag Package differs from the previous approach by implementing new features and support for additional irrigation providers. New features include representation of deficit and over-irrigation, Multi-Aquifer Well and Lake Package irrigation providers, and support for structured, vertex, and unstructured grid models. Three example problems are presented that demonstrate how the API-Ag Package improves representation of highly managed systems and are further used to validate the irrigation demand and delivery formulations. Irrigation volumes simulated in the three example problems show excellent agreement with the MODFLOW-NWT AG Package.

FloPy Workflows for Creating Structured and Unstructured MODFLOW Models.

FloPy is a Python package for creating, running, and post-processing MODFLOW-based groundwater flow and transport models. FloPy functionality has expanded to support the latest version of MODFLOW (MODFLOW 6) including support for unstructured grids. FloPy can simplify the process required to download MODFLOW-based and other executables for Linux, MacOS, and Windows operating systems. Expanded FloPy capabilities include (1) full support for structured and unstructured spatial discretizations; (2) geoprocessing of spatial features and raster data to develop model input for supported discretization types; (3) the addition of functionality to provide direct access to simulated output data; (4) extension of plotting capabilities to unstructured MODFLOW 6 discretization types; and (5) the ability to export model data to shapefiles, NetCDF, and VTK formats for processing, analysis, and visualization by other software products. Examples of using expanded FloPy capabilities are presented for a hypothetical watershed. An unstructured groundwater flow and transport model, with several advanced stress packages, is presented to demonstrate how FloPy can be used to develop complicated unstructured model datasets from original source data (shapefiles and rasters), post-process model results, and plot simulated results.

Genome sequence of white sturgeon herpesvirus 1 isolated from farmed white sturgeon (Acipenser transmontanus).

The genome sequence of white sturgeon herpesvirus 1, which was isolated from farmed white sturgeon (Acipenser transmontanus), was determined. Comparative analyses suggest the classification of this virus as a new species in a new genus in the family Alloherpesviridae.

Resurrection of 2'-5'-oligoadenylate synthetase 1 (OAS1) from the ancestor of modern horseshoe bats blocks SARS-CoV-2 replication.

The prenylated form of the human 2'-5'-oligoadenylate synthetase 1 (OAS1) protein has been shown to potently inhibit the replication of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. However, the OAS1 orthologue in the horseshoe bats (superfamily Rhinolophoidea), the reservoir host of SARS-related coronaviruses (SARSr-CoVs), has lost the prenylation signal required for this antiviral activity. Herein, we used an ancestral state reconstruction approach to predict and reconstitute in vitro, the most likely OAS1 protein sequence expressed by the Rhinolophoidea common ancestor prior to its prenylation loss (RhinoCA OAS1). We exogenously expressed the ancient bat protein in vitro to show that, unlike its non-prenylated horseshoe bat descendants, RhinoCA OAS1 successfully blocks SARS-CoV-2 replication. Using protein structure predictions in combination with evolutionary hypothesis testing methods, we highlight sites under unique diversifying selection specific to OAS1's evolution in the Rhinolophoidea. These sites are located near the RNA-binding region and the C-terminal end of the protein where the prenylation signal would have been. Our results confirm that OAS1 prenylation loss at the base of the Rhinolophoidea clade ablated the ability of OAS1 to restrict SARSr-CoV replication and that subsequent evolution of the gene in these bats likely favoured an alternative function. These findings can advance our understanding of the tightly linked association between SARSr-CoVs and horseshoe bats.

HCMV-secreted glycoprotein gpUL4 inhibits TRAIL-mediated apoptosis and NK cell activation.

Human cytomegalovirus (HCMV) is a paradigm of pathogen immune evasion and sustains lifelong persistent infection in the face of exceptionally powerful host immune responses through the concerted action of multiple immune-evasins. These reduce NK cell activation by inhibiting ligands for activating receptors, expressing ligands for inhibitory receptors, or inhibiting synapse formation. However, these functions only inhibit direct interactions with the infected cell. To determine whether the virus also expresses soluble factors that could modulate NK function at a distance, we systematically screened all 170 HCMV canonical protein-coding genes. This revealed that UL4 encodes a secreted and heavily glycosylated protein (gpUL4) that is expressed with late-phase kinetics and is capable of inhibiting NK cell degranulation. Analyses of gpUL4 binding partners by mass spectrometry identified an interaction with TRAIL. gpUL4 bound TRAIL with picomolar affinity and prevented TRAIL from binding its receptor, thus acting as a TRAIL decoy receptor. TRAIL is found in both soluble and membrane-bound forms, with expression of the membrane-bound form strongly up-regulated on NK cells in response to interferon. gpUL4 inhibited apoptosis induced by soluble TRAIL, while also binding to the NK cell surface in a TRAIL-dependent manner, where it blocked NK cell degranulation and cytokine secretion. gpUL4 therefore acts as an immune-evasin by inhibiting both soluble and membrane-bound TRAIL and is a viral-encoded TRAIL decoy receptor. Interestingly, gpUL4 could also suppress NK responses to heterologous viruses, suggesting that it may act as a systemic virally encoded immunosuppressive agent.

The SARS-CoV-2 Spike Protein Mutation Explorer: using an interactive application to improve the public understanding of SARS-CoV-2 variants of concern.

Due to the COVID-19 pandemic the virus responsible, SARS-CoV-2, became a source of intense interest for non-expert audiences. The viral spike protein gained particular public interest as the main target for protective immune responses, including those elicited by vaccines. The rapid evolution of SARS-CoV-2 resulted in variations in the spike that enhanced transmissibility or weakened vaccine protection. This created new variants of concern (VOCs). The emergence of VOCs was studied using viral sequence data which was shared through portals such as the online Mutation Explorer of the COVID-19 Genomics UK consortium (COG-UK/ME). This was designed for an expert audience, but the information it contained could be of general interest if suitably communicated. Visualisations, interactivity and animation can improve engagement and understanding of molecular biology topics, and so we developed a graphical educational resource, the SARS-CoV-2 Spike Protein Mutation Explorer (SSPME), which used interactive 3D molecular models and animations to explain the molecular biology underpinning VOCs. User testing showed that the SSPME had better usability and improved participant knowledge confidence and knowledge acquisition compared to COG-UK/ME. This demonstrates how interactive visualisations can be used for effective molecular biology communication, as well as improving the public understanding of SARS-CoV-2 VOCs.

Isolation and genome sequencing of cytomegaloviruses from Natal multimammate mice (Mastomys natalensis).

Distinct cytomegaloviruses (CMVs) are widely distributed across their mammalian hosts in a highly host species-restricted pattern. To date, evidence demonstrating this has been limited largely to PCR-based approaches targeting small, conserved genomic regions, and only a few complete genomes of isolated viruses representing distinct CMV species have been sequenced. We have now combined direct isolation of infectious viruses from tissues with complete genome sequencing to provide a view of CMV diversity in a wild animal population. We targeted Natal multimammate mice (Mastomys natalensis), which are common in sub-Saharan Africa, are known to carry a variety of zoonotic pathogens, and are regarded as the primary source of Lassa virus (LASV) spillover into humans. Using transformed epithelial cells prepared from M. natalensis kidneys, we isolated CMVs from the salivary gland tissue of 14 of 37 (36 %) animals from a field study site in Mali. Genome sequencing showed that these primary isolates represent three different M. natalensis CMVs (MnatCMVs: MnatCMV1, MnatCMV2 and MnatCMV3), with some animals carrying multiple MnatCMVs or multiple strains of a single MnatCMV presumably as a result of coinfection or superinfection. Including primary isolates and plaque-purified isolates, we sequenced and annotated the genomes of two MnatCMV1 strains (derived from sequencing 14 viruses), six MnatCMV2 strains (25 viruses) and ten MnatCMV3 strains (21 viruses), totalling 18 MnatCMV strains isolated as 60 infectious viruses. Phylogenetic analysis showed that these MnatCMVs group with other murid viruses in the genus Muromegalovirus (subfamily Betaherpesvirinae, family Orthoherpesviridae), and that MnatCMV1 and MnatCMV2 are more closely related to each other than to MnatCMV3. The availability of MnatCMV isolates and the characterization of their genomes will serve as the prelude to the generation of a MnatCMV-based vaccine to target LASV in the M. natalensis reservoir.

The recency and geographical origins of the bat viruses ancestral to SARS-CoV and SARS-CoV-2.

The emergence of SARS-CoV in 2002 and SARS-CoV-2 in 2019 has led to increased sampling of related sarbecoviruses circulating primarily in horseshoe bats. These viruses undergo frequent recombination and exhibit spatial structuring across Asia. Employing recombination-aware phylogenetic inference on bat sarbecoviruses, we find that the closest-inferred bat virus ancestors of SARS-CoV and SARS-CoV-2 existed just ~1-3 years prior to their emergence in humans. Phylogeographic analyses examining the movement of related sarbecoviruses demonstrate that they traveled at similar rates to their horseshoe bat hosts and have been circulating for thousands of years in Asia. The closest-inferred bat virus ancestor of SARS-CoV likely circulated in western China, and that of SARS-CoV-2 likely circulated in a region comprising southwest China and northern Laos, both a substantial distance from where they emerged. This distance and recency indicate that the direct ancestors of SARS-CoV and SARS-CoV-2 could not have reached their respective sites of emergence via the bat reservoir alone. Our recombination-aware dating and phylogeographic analyses reveal a more accurate inference of evolutionary history than performing only whole-genome or single gene analyses. These results can guide future sampling efforts and demonstrate that viral genomic fragments extremely closely related to SARS-CoV and SARS-CoV-2 were circulating in horseshoe bats, confirming their importance as the reservoir species for SARS viruses.

BTN3A3 evasion promotes the zoonotic potential of influenza A viruses.

Spillover events of avian influenza A viruses (IAVs) to humans could represent the first step in a future pandemic1. Several factors that limit the transmission and replication of avian IAVs in mammals have been identified. There are several gaps in our understanding to predict which virus lineages are more likely to cross the species barrier and cause disease in humans1. Here, we identified human BTN3A3 (butyrophilin subfamily 3 member A3)2 as a potent inhibitor of avian IAVs but not human IAVs. We determined that BTN3A3 is expressed in human airways and its antiviral activity evolved in primates. We show that BTN3A3 restriction acts primarily at the early stages of the virus life cycle by inhibiting avian IAV RNA replication. We identified residue 313 in the viral nucleoprotein (NP) as the genetic determinant of BTN3A3 sensitivity (313F or, rarely, 313L in avian viruses) or evasion (313Y or 313V in human viruses). However, avian IAV serotypes, such as H7 and H9, that spilled over into humans also evade BTN3A3 restriction. In these cases, BTN3A3 evasion is due to substitutions (N, H or Q) in NP residue 52 that is adjacent to residue 313 in the NP structure3. Thus, sensitivity or resistance to BTN3A3 is another factor to consider in the risk assessment of the zoonotic potential of avian influenza viruses.

Identifying potential significant factors impacting zero-inflated proportion data.

Classical supervised methods like linear regression and decision trees are not completely adapted for identifying impacting factors on a response variable corresponding to zero-inflated proportion data (ZIPD) that are dependent, continuous and bounded. In this article we propose a within-block permutation-based methodology to identify factors (discrete or continuous) that are significantly correlated with ZIPD, we propose a performance indicator quantifying the percentage of correlation explained by the subset of significant factors, and we show how to predict the ranks of the response variables conditionally on the observation of these factors. The methodology is illustrated on simulated data and on two real data sets dealing with epidemiology. In the first data set, ZIPD correspond to probabilities of transmission of Influenza between horses. In the second data set, ZIPD correspond to probabilities that geographic entities (eg, states and countries) have the same COVID-19 mortality dynamics.

The Disproportionate Impact of Fear of Negative Evaluation on First-Generation College Students, LGBTQ+ Students, and Students with Disabilities in College Science Courses.

Fear of negative evaluation (FNE), defined as a sense of dread associated with being negatively judged in a social situation, has been identified as the primary factor underlying undergraduate anxiety in active-learning science courses. However, no quantitative studies have examined the extent to which science undergraduates experience FNE and how they are impacted by FNE in college science courses. To address this gap, we surveyed 566 undergraduates from one university in the U.S. Southwest who were enrolled in life sciences courses where they had opportunities to speak in front of the whole class. Participants were asked a suite of questions regarding their experiences with FNE in large-enrollment college science courses. We found that first-generation college students, LGBTQ+ students, and students with disabilities reported disproportionately high levels of FNE compared with their counterparts. Additionally, students reported that FNE can cause them to overthink their responses and participate less in class. Participants rated being cold called and presenting alone as forms of whole-class participation that elicit the highest levels of FNE. This research highlights the impact of FNE on undergraduates and provides student-generated recommendations to reduce FNE in active-learning science courses.

Directions of change in intrinsic case severity across successive SARS-CoV-2 variant waves have been inconsistent.

OBJECTIVES: To determine how the intrinsic severity of successively dominant SARS-CoV-2 variants changed over the course of the pandemic. METHODS: A retrospective cohort analysis in the NHS Greater Glasgow and Clyde (NHS GGC) Health Board. All sequenced non-nosocomial adult COVID-19 cases in NHS GGC with relevant SARS-CoV-2 lineages (B.1.177/Alpha, Alpha/Delta, AY.4.2 Delta/non-AY.4.2 Delta, non-AY.4.2 Delta/Omicron, and BA.1 Omicron/BA.2 Omicron) during analysis periods were included. Outcome measures were hospital admission, ICU admission, or death within 28 days of positive COVID-19 test. We report the cumulative odds ratio; the ratio of the odds that an individual experiences a severity event of a given level vs all lower severity levels for the resident and the replacement variant after adjustment. RESULTS: After adjustment for covariates, the cumulative odds ratio was 1.51 (95% CI: 1.08-2.11) for Alpha versus B.1.177, 2.09 (95% CI: 1.42-3.08) for Delta versus Alpha, 0.99 (95% CI: 0.76-1.27) for AY.4.2 Delta versus non-AY.4.2 Delta, 0.49 (95% CI: 0.22-1.06) for Omicron versus non-AY.4.2 Delta, and 0.86 (95% CI: 0.68-1.09) for BA.2 Omicron versus BA.1 Omicron. CONCLUSIONS: The direction of change in intrinsic severity between successively emerging SARS-CoV-2 variants was inconsistent, reminding us that the intrinsic severity of future SARS-CoV-2 variants remains uncertain.

The SARS-CoV-2 Alpha variant was associated with increased clinical severity of COVID-19 in Scotland: A genomics-based retrospective cohort analysis.

OBJECTIVES: The SARS-CoV-2 Alpha variant was associated with increased transmission relative to other variants present at the time of its emergence and several studies have shown an association between Alpha variant infection and increased hospitalisation and 28-day mortality. However, none have addressed the impact on maximum severity of illness in the general population classified by the level of respiratory support required, or death. We aimed to do this. METHODS: In this retrospective multi-centre clinical cohort sub-study of the COG-UK consortium, 1475 samples from Scottish hospitalised and community cases collected between 1st November 2020 and 30th January 2021 were sequenced. We matched sequence data to clinical outcomes as the Alpha variant became dominant in Scotland and modelled the association between Alpha variant infection and severe disease using a 4-point scale of maximum severity by 28 days: 1. no respiratory support, 2. supplemental oxygen, 3. ventilation and 4. death. RESULTS: Our cumulative generalised linear mixed model analyses found evidence (cumulative odds ratio: 1.40, 95% CI: 1.02, 1.93) of a positive association between increased clinical severity and lineage (Alpha variant versus pre-Alpha variants). CONCLUSIONS: The Alpha variant was associated with more severe clinical disease in the Scottish population than co-circulating lineages.