Sequencing and Analysis of Lumpy Skin Disease Virus Whole Genomes Reveals a New Viral Subgroup in West and Central Africa.

Lumpy skin disease virus (LSDV) is a member of the capripoxvirus (CPPV) genus of the Poxviridae family. LSDV is a rapidly emerging, high-consequence pathogen of cattle, recently spreading from Africa and the Middle East into Europe and Asia. We have sequenced the whole genome of historical LSDV isolates from the Pirbright Institute virus archive, and field isolates from recent disease outbreaks in Sri Lanka, Mongolia, Nigeria and Ethiopia. These genome sequences were compared to published genomes and classified into different subgroups. Two subgroups contained vaccine or vaccine-like samples ("Neethling-like" clade 1.1 and "Kenya-like" subgroup, clade 1.2.2). One subgroup was associated with outbreaks of LSD in the Middle East/Europe (clade 1.2.1) and a previously unreported subgroup originated from cases of LSD in west and central Africa (clade 1.2.3). Isolates were also identified that contained a mix of genes from both wildtype and vaccine samples (vaccine-like recombinants, grouped in clade 2). Whole genome sequencing and analysis of LSDV strains isolated from different regions of Africa, Europe and Asia have provided new knowledge of the drivers of LSDV emergence, and will inform future disease control strategies.

West Nile virus spread in Europe: Phylogeographic pattern analysis and key drivers.

BACKGROUND: West Nile virus (WNV) outbreaks in birds, humans, and livestock have occurred in multiple areas in Europe and have had a significant impact on animal and human health. The patterns of emergence and spread of WNV in Europe are very different from those in the US and understanding these are important for guiding preparedness activities. METHODS: We mapped the evolution and spread history of WNV in Europe by incorporating viral genome sequences and epidemiological data into phylodynamic models. Spatially explicit phylogeographic models were developed to explore the possible contribution of different drivers to viral dispersal direction and velocity. A "skygrid-GLM" approach was used to identify how changes in environments would predict viral genetic diversity variations over time. FINDINGS: Among the six lineages found in Europe, WNV-2a (a sub-lineage of WNV-2) has been predominant (accounting for 73% of all sequences obtained in Europe that have been shared in the public domain) and has spread to at least 14 countries. In the past two decades, WNV-2a has evolved into two major co-circulating clusters, both originating from Central Europe, but with distinct dynamic history and transmission patterns. WNV-2a spreads at a high dispersal velocity (88km/yr-215 km/yr) which is correlated to bird movements. Notably, amongst multiple drivers that could affect the spread of WNV, factors related to land use were found to strongly influence the spread of WNV. Specifically, the intensity of agricultural activities (defined by factors related to crops and livestock production, such as coverage of cropland, pasture, cultivated and managed vegetation, livestock density) were positively associated with both spread direction and velocity. In addition, WNV spread direction was associated with high coverage of wetlands and migratory bird flyways. CONCLUSION: Our results suggest that-in addition to ecological conditions favouring bird- and mosquito- presence-agricultural land use may be a significant driver of WNV emergence and spread. Our study also identified significant gaps in data and the need to strengthen virological surveillance in countries of Central Europe from where WNV outbreaks are likely seeded. Enhanced monitoring for early detection of further dispersal could be targeted to areas with high agricultural activities and habitats of migratory birds.

Defining the pig microglial transcriptome reveals its core signature, regional heterogeneity, and similarity with human and rodent microglia.

Microglia play key roles in brain homeostasis as well as responses to neurodegeneration and neuroinflammatory processes caused by physical disease and psychosocial stress. The pig is a physiologically relevant model species for studying human neurological disorders, many of which are associated with microglial dysfunction. Furthermore, pigs are an important agricultural species, and there is a need to understand how microglial function affects their welfare. As a basis for improved understanding to enhance biomedical and agricultural research, we sought to characterize pig microglial identity at genome-wide scale and conduct inter-species comparisons. We isolated pig hippocampal tissue and microglia from frontal cortex, hippocampus, and cerebellum, as well as alveolar macrophages from the lungs and conducted RNA-sequencing (RNAseq). By comparing the transcriptomic profiles between microglia, macrophages, and hippocampal tissue, we derived a set of 239 highly enriched genes defining the porcine core microglial signature. We found brain regional heterogeneity based on 150 genes showing significant (adjusted p < 0.01) regional variations and that cerebellar microglia were most distinct. We compared normalized gene expression for microglia from human, mice and pigs using microglia signature gene lists derived from each species and demonstrated that a core microglial marker gene signature is conserved across species, but that species-specific expression subsets also exist. Our data provide a valuable resource defining the pig microglial transcriptome signature that validates and highlights pigs as a useful large animal species bridging between rodents and humans in which to study the role of microglia during homeostasis and disease.

Graphia: A platform for the graph-based visualisation and analysis of high dimensional data.

Graphia is an open-source platform created for the graph-based analysis of the huge amounts of quantitative and qualitative data currently being generated from the study of genomes, genes, proteins metabolites and cells. Core to Graphia's functionality is support for the calculation of correlation matrices from any tabular matrix of continuous or discrete values, whereupon the software is designed to rapidly visualise the often very large graphs that result in 2D or 3D space. Following graph construction, an extensive range of measurement algorithms, routines for graph transformation, and options for the visualisation of node and edge attributes are available, for graph exploration and analysis. Combined, these provide a powerful solution for the interpretation of high-dimensional data from many sources, or data already in the form of a network or equivalent adjacency matrix. Several use cases of Graphia are described, to showcase its wide range of applications in the analysis biological data. Graphia runs on all major desktop operating systems, is extensible through the deployment of plugins and is freely available to download from https://graphia.app/.

Aging-Related Impairments to M Cells in Peyer's Patches Coincide With Disturbances to Paneth Cells.

The decline in mucosal immunity during aging increases susceptibility, morbidity and mortality to infections acquired via the gastrointestinal and respiratory tracts in the elderly. We previously showed that this immunosenescence includes a reduction in the functional maturation of M cells in the follicle-associated epithelia (FAE) covering the Peyer's patches, diminishing the ability to sample of antigens and pathogens from the gut lumen. Here, co-expression analysis of mRNA-seq data sets revealed a general down-regulation of most FAE- and M cell-related genes in Peyer's patches from aged mice, including key transcription factors known to be essential for M cell differentiation. Conversely, expression of ACE2, the cellular receptor for SARS-Cov-2 virus, was increased in the aged FAE. This raises the possibility that the susceptibility of aged Peyer's patches to infection with the SARS-Cov-2 virus is increased. Expression of key Paneth cell-related genes was also reduced in the ileum of aged mice, consistent with the adverse effects of aging on their function. However, the increased expression of these genes in the villous epithelium of aged mice suggested a disturbed distribution of Paneth cells in the aged intestine. Aging effects on Paneth cells negatively impact on the regenerative ability of the gut epithelium and could indirectly impede M cell differentiation. Thus, restoring Paneth cell function may represent a novel means to improve M cell differentiation in the aging intestine and increase mucosal vaccination efficacy in the elderly.

Vitamin D3 replacement enhances antigen-specific immunity in older adults.

Introduction: Ageing is associated with increased number of infections, decreased vaccine efficacy and increased systemic inflammation termed inflammageing. These changes are reflected by reduced recall responses to varicella zoster virus (VZV) challenge in the skin of older adults. Vitamin D deficiency is more common in the old and has been associated with frailty and increased inflammation. In addition, vitamin D increases immunoregulatory mechanisms and therefore has the potential to inhibit inflammageing. Objectives: We investigated the use of vitamin D3 replacement to enhance cutaneous antigen-specific immunity in older adults (≥65 years). Methods: Vitamin D insufficient older adults (n = 18) were administered 6400IU of vitamin D3/day orally for 14 weeks. Antigen-specific immunity to VZV was assessed by clinical score assessment of the injection site and transcriptional analysis of skin biopsies collected from challenged injection sites pre- and post-vitamin D3 replacement. Results: We showed that older adults had reduced VZV-specific cutaneous immune response and increased non-specific inflammation as compared to young. Increased non-specific inflammation observed in the skin of older adults negatively correlated with vitamin D sufficiency. We showed that vitamin D3 supplementation significantly increased the response to cutaneous VZV antigen challenge in older adults. This enhancement was associated with a reduction in inflammatory monocyte infiltration with a concomitant enhancement of T cell recruitment to the site of antigen challenge in the skin. Conclusion: Vitamin D3 replacement can boost antigen-specific immunity in older adults with sub-optimal vitamin D status.

Recruitment of inflammatory monocytes by senescent fibroblasts inhibits antigen-specific tissue immunity during human aging.

We have previously shown that healthy older adults exhibit reduced cutaneous immune responses during a varicella zoster virus (VZV) antigen challenge that correlated with a nonspecific inflammatory response to the injection itself. Here we found that needle damage during intradermal injections in older adults led to an increase in the number of cutaneous senescent fibroblasts expressing CCL2, resulting in the local recruitment of inflammatory monocytes. These infiltrating monocytes secreted prostaglandin E2, which inhibited resident memory T cell activation and proliferation. Pretreatment of older participants with a p38 mitogen-activated protein kinase inhibitor in vivo decreased CCL2 expression and inhibited monocyte recruitment and secretion of prostaglandin E2. This coincided with an increased response to VZV antigen challenge in the skin. Our results point to a series of molecular and cellular mechanisms that link cellular senescence, tissue damage, excessive inflammation and reduced immune responsiveness in human skin and demonstrate that tissue-specific immunity can be restored in older adults by short-term inhibition of inflammatory responses.

Influence of skin melanisation and ultraviolet radiation on biomarkers of systemic oxidative stress.

Skin melanisation ranges widely across human populations. Melanin has antioxidant properties and also acts as a filter to solar ultraviolet radiation (UVR) incident upon the skin. In this study we firstly examined whether melanin level might influence baseline levels of systemic oxidative stress, in 65 humans in vivo from the same geographical area ranging from the lightest to darkest skin type (phototype I-VI). This was examined in winter-time (latitude 53.5°N). Remarkably, we found that urinary biomarkers of oxidatively-generated DNA damage (8-oxodG) and RNA damage (8-oxoGuo) were significantly correlated with skin lightness (L*), such that 14-15% of the variation in their baseline levels could be explained by skin colour. Next we exposed 15 humans at the extremes of skin melanisation to a simulated summer-time exposure of solar UVR (95% UVA, 5% UVB; dose standardised to sunburn threshold), following which they provided a sample of every urine void over the next five days. We found that UVR induced a small but significant increase in urinary 8-oxodG and 8-oxoGuo, with differing kinetics between skin types. Thus greater melanisation is associated with protection against systemic oxidative stress, which may reflect melanin's antioxidant properties, and solar UVR exposure also influences systemic oxidative stress levels in humans. These novel findings may have profound implications for human physiology and health.

Computational Analysis of SARS-CoV-2 and SARS-Like Coronavirus Diversity in Human, Bat and Pangolin Populations.

In 2019, a novel coronavirus, SARS-CoV-2/nCoV-19, emerged in Wuhan, China, and has been responsible for the current COVID-19 pandemic. The evolutionary origins of the virus remain elusive and understanding its complex mutational signatures could guide vaccine design and development. As part of the international "CoronaHack" in April 2020, we employed a collection of contemporary methodologies to compare the genomic sequences of coronaviruses isolated from human (SARS-CoV-2; n = 163), bat (bat-CoV; n = 215) and pangolin (pangolin-CoV; n = 7) available in public repositories. We have also noted the pangolin-CoV isolate MP789 to bare stronger resemblance to SARS-CoV-2 than other pangolin-CoV. Following de novo gene annotation prediction, analyses of gene-gene similarity network, codon usage bias and variant discovery were undertaken. Strong host-associated divergences were noted in ORF3a, ORF6, ORF7a, ORF8 and S, and in codon usage bias profiles. Last, we have characterised several high impact variants (in-frame insertion/deletion or stop gain) in bat-CoV and pangolin-CoV populations, some of which are found in the same amino acid position and may be highlighting loci of potential functional relevance.

Immune Cell Gene Signatures for Profiling the Microenvironment of Solid Tumors.

The immune composition of the tumor microenvironment regulates processes including angiogenesis, metastasis, and the response to drugs or immunotherapy. To facilitate the characterization of the immune component of tumors from transcriptomics data, a number of immune cell transcriptome signatures have been reported that are made up of lists of marker genes indicative of the presence a given immune cell population. The majority of these gene signatures have been defined through analysis of isolated blood cells. However, blood cells do not reflect the differentiation or activation state of similar cells within tissues, including tumors, and consequently markers derived from blood cells do not necessarily transfer well to tissues. To address this issue, we generated a set of immune gene signatures derived directly from tissue transcriptomics data using a network-based deconvolution approach. We define markers for seven immune cell types, collectively named ImSig, and demonstrate how these markers can be used for the quantitative estimation of the immune cell content of tumor and nontumor tissue samples. The utility of ImSig is demonstrated through the stratification of melanoma patients into subgroups of prognostic significance and the identification of immune cells with the use of single-cell RNA-sequencing data derived from tumors. Use of ImSig is facilitated by an R package (imsig). Cancer Immunol Res; 6(11); 1388-400. ©2018 AACR.

Fractional Sunburn Threshold UVR Doses Generate Equivalent Vitamin D and DNA Damage in Skin Types I-VI but with Epidermal DNA Damage Gradient Correlated to Skin Darkness.

Public health guidance recommends limiting sun exposure to sub-sunburn levels, but it is unknown whether these can gain vitamin D (for musculoskeletal health) while avoiding epidermal DNA damage (initiates skin cancer). Well-characterized healthy humans of all skin types (I-VI, lightest to darkest skin) were exposed to a low-dose series of solar simulated UVR of 20%-80% their individual sunburn threshold dose (minimal erythema dose). Significant UVR dose responses were seen for serum 25-hydroxyvitamin D and whole epidermal cyclobutane pyrimidine dimers (CPDs), with as little as 0.2 minimal erythema dose concurrently producing 25-hydroxyvitamin D and CPD. Fractional MEDs generated equivalent levels of whole epidermal CPD and 25-hydroxyvitamin D across all skin types. Crucially, we showed an epidermal gradient of CPD formation strongly correlated with skin darkness (r = 0.74, P < 0.0001), which reflected melanin content and showed increasing protection across the skin types, ranging from darkest skin, where high CPD levels occurred superficially, with none in the germinative basal layer, to lightest skin, where CPD levels were induced evenly across the epidermal depth. People with darker skin can be encouraged to use sub-sunburn UVR-exposure to enhance their vitamin D. In people with lighter skin, basal cell damage occurs concurrent with vitamin D synthesis at exquisitely low UVR levels, providing an explanation for their high skin cancer incidence; greater caution is required.

Derivation of marker gene signatures from human skin and their use in the interpretation of the transcriptional changes associated with dermatological disorders.

Numerous studies have explored the altered transcriptional landscape associated with skin diseases to understand the nature of these disorders. However, data interpretation represents a significant challenge due to a lack of good maker sets for many of the specialized cell types that make up this tissue, whose composition may fundamentally alter during disease. Here we have sought to derive expression signatures that define the various cell types and structures that make up human skin, and demonstrate how they can be used to aid the interpretation of transcriptomic data derived from this organ. Two large normal skin transcriptomic datasets were identified, one RNA-seq (n = 578), the other microarray (n = 165), quality controlled and subjected separately to network-based analyses to identify clusters of robustly co-expressed genes. The biological significance of these clusters was then assigned using a combination of bioinformatics analyses, literature, and expert review. After cross comparison between analyses, 20 gene signatures were defined. These included expression signatures for hair follicles, glands (sebaceous, sweat, apocrine), keratinocytes, melanocytes, endothelia, muscle, adipocytes, immune cells, and a number of pathway systems. Collectively, we have named this resource SkinSig. SkinSig was then used in the analysis of transcriptomic datasets for 18 skin conditions, providing in-context interpretation of these data. For instance, conventional analysis has shown there to be a decrease in keratinization and fatty metabolism with age; we more accurately define these changes to be due to loss of hair follicles and sebaceous glands. SkinSig also highlighted the over-/under-representation of various cell types in skin diseases, reflecting an influx in immune cells in inflammatory disorders and a relative reduction in other cell types. Overall, our analyses demonstrate the value of this new resource in defining the functional profile of skin cell types and appendages, and in improving the interpretation of disease data. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Genome-wide high-resolution screening in Dupuytren's disease reveals common regions of DNA copy number alterations.

PURPOSE: Dupuytren's disease (DD) is a familial disorder with a high genetic susceptibility in white people; however, its etiopathogenesis remains unknown. Previous comparative genomic hybridization studies using lower-resolution, 44-k oligonucleotide-based arrays revealed no copy number variation (CNV) changes in DD. In this study, we used a higher-resolution genome-wide screening (next-generation microarrays) comprising 963,331 human sequences (3 kb spacing between probes) for whole genome DNA variation analysis. The objective was to detect cryptic chromosomal imbalances in DD. METHODS: Agilent SurePrint G3 microarrays, one million format (Agilent Technologies, Santa Clara, CA), were used to detect CNV regions (CNVRs) in DNA extracted from nodules of 4 white men with DD (age, 69 +/- 4 y). Reference samples were from the DNA of 10 men who served as control patients. Copy number variations that were common to greater than 3 assessed DD individuals (p < .05) were selected as candidate loci for DD etiology. In addition, quantitative polymerase chain reactions (qPCR) assays were designed for selected CNVRs on DNA from 13 DD patients and 11 control patients. Independent t-tests and Fisher's exact tests were carried out for statistical analysis. RESULTS: Three novel CNVs previously unreported in the phenotypically normal population were detected in 3 DD cases, located at 10q22, 16p12.1, and 17p12. Nine polymorphic CNVRs potentially associated with DD were determined using our strategic selection criteria, locating to chromosomes 1q31, 6p21, 7p14, 8p11, 12p13, 14q11, 17q21 and 20p13. More than 3 of the DD cases tested had a CNVR located to a small region on 6p21 and 4 CNVRs within 6p21-22 of the human leukocyte antigen (HLA) genes. CONCLUSIONS: Three novel copy number alterations were observed in 3 unrelated patients with sporadic (no known family history) DD. Nine polymorphic CNVRs were found to be common among the DD cases. These variants might contain genes involved in DD formation, indicating that important gene networks expressed within the palmar fascia might contribute to genetic susceptibility of DD.