Squirrelpox virus: assessing prevalence, transmission and environmental degradation

Red squirrels (Sciurus vulgaris) declined in Great Britain and Ireland during the last century, due to habitat loss and the introduction of grey squirrels (Sciurus carolinensis), which competitively exclude the red squirrel and act as a reservoir for squirrelpox virus (SQPV). The disease is generally fatal to red squirrels and their ecological replacement by grey squirrels is up to 25 times faster where the virus is present. We aimed to determine: (1) the seropositivity and prevalence of SQPV DNA in the invasive and native species at a regional scale; (2) possible SQPV transmission routes; and, (3) virus degradation rates under differing environmental conditions. Grey (n = 208) and red (n = 40) squirrel blood and tissues were sampled. Enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qPCR) techniques established seropositivity and viral DNA presence, respectively. Overall 8% of squirrels sampled (both species combined) had evidence of SQPV DNA in their tissues and 22% were in possession of antibodies. SQPV prevalence in sampled red squirrels was 2.5%. Viral loads were typically low in grey squirrels by comparison to red squirrels. There was a trend for a greater number of positive samples in spring and summer than in winter. Possible transmission routes were identified through the presence of viral DNA in faeces (red squirrels only), urine and ectoparasites (both species). Virus degradation analyses suggested that, after 30 days of exposure to six combinations of environments, there were more intact virus particles in scabs kept in warm (25°C) and dry conditions than in cooler (5 and 15°C) or wet conditions. We conclude that SQPV is present at low prevalence in invasive grey squirrel populations with a lower prevalence in native red squirrels. Virus transmission could occur through urine especially during warm dry summer conditions but, more notably, via ectoparasites, which are shared by both species

Herpesvirus skin disease in free-living common frogs Rana temporaria in Great Britain

Infectious disease is a significant driver of global amphibian declines, yet despite this, relatively little is known about the range of pathogens that affect free-living amphibians. Recent detection of the tentatively named Ranid herpesvirus 3 (RHV3), associated with skin disease in free-living common frogs Rana temporaria in Switzerland, helps to address this paucity in knowledge, but the geographic distribution and epidemiology of the pathogen remains unclear. Syndromic surveillance for ranid herpesvirus skin disease was undertaken throughout Great Britain (GB), January 2014 to December 2016. Reports of common frogs with macroscopic skin lesions with a characteristic grey appearance were solicited from members of the public. Post-mortem examination was conducted on one affected frog found dead in 2015 at a site in England. In addition, archived samples from an incident involving common frogs in England in 1997 with similar macroscopic lesions were further investigated. Transmission electron microscopy identified herpes-like virions in skin lesions from both the 1997 and 2015 incidents. RHV3, or RHV3-like virus, was detected in skin lesions from the 2015 case by PCR and sequencing. Our findings indicate that herpesvirus skin disease is endemic in common frogs in GB, with widespread distribution at apparently low prevalence. Further research into the role of host immunity, virus latency and the significance of infection to host survival is required to better understand the epidemiology and impact of cutaneous herpesvirus infections in amphibian populations

Analysis of Campylobacter jejuni infection in the gnotobiotic piglet and genome-wide identification of bacterial factors required for infection

To investigate how Campylobacter jejuni causes the clinical symptoms of diarrhoeal disease in humans, use of a relevant animal model is essential. Such a model should mimic the human disease closely in terms of host physiology, incubation period before onset of disease, clinical signs and a comparable outcome of disease. In this study, we used a gnotobiotic piglet model to study determinants of pathogenicity of C. jejuni. In this model, C. jejuni successfully established infection and piglets developed an increased temperature with watery diarrhoea, which was caused by a leaky epithelium and reduced bile re-absorption in the intestines. Further, we assessed the C. jejuni genes required for infection of the porcine gastrointestinal tract utilising a transposon (Tn) mutant library screen. A total of 123 genes of which Tn mutants showed attenuated piglet infection were identified. Our screen highlighted a crucial role for motility and chemotaxis, as well as central metabolism. In addition, Tn mutants of 14 genes displayed enhanced piglet infection. This study gives a unique insight into the mechanisms of C. jejuni disease in terms of host physiology and contributing bacterial factors.This work was funded by BBSRC awards BB/F020988/1 and BB/K004514/1. Moredun Research Institute receives funding via Rural and Environment Science and Analytical Services (RESAS) division of the Scottish Government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Unexpected differential metabolic responses of Campylobacter jejuni to the abundant presence of glutamate and fucose

Introduction: Campylobacter jejuni is the leading cause of foodborne bacterial enteritis in humans, and yet little is known in regard to how genetic diversity and metabolic capabilities among isolates affect their metabolic phenotype and pathogenicity. Objectives: For instance, the C. jejuni 11168 strain can utilize both l-fucose and l-glutamate as a carbon source, which provides the strain with a competitive advantage in some environments and in this study we set out to assess the metabolic response of C. jejuni 11168 to the presence of l-fucose and l-glutamate in the growth medium. Methods: To achieve this, untargeted hydrophilic liquid chromatography coupled to mass spectrometry was used to obtain metabolite profiles of supernatant extracts obtained at three different time points up to 24 h. Results: This study identified both the depletion and the production and subsequent release of a multitude of expected and unexpected metabolites during the growth of C. jejuni 11168 under three different conditions. A large set of standards allowed identification of a number of metabolites. Further mass spectrometry fragmentation analysis allowed the additional annotation of substrate-specific metabolites. The results show that C. jejuni 11168 upon l-fucose addition indeed produces degradation products of the fucose pathway. Furthermore, methionine was faster depleted from the medium, consistent with previously-observed methionine auxotrophy. Conclusions: Moreover, a multitude of not previously annotated metabolites in C. jejuni were found to be increased specifically upon l-fucose addition. These metabolites may well play a role in the pathogenicity of this C. jejuni strain.</p

Limited diversity associated with duplicated class II MHC-DRB genes in the red squirrel population in the United Kingdom compared with continental Europe

The red squirrel (Sciurus vulgaris) population in the United Kingdom has declined over the last century and is now on the UK endangered species list. This is the result of competition from the eastern grey squirrel (S. carolinensis) which was introduced in the 19th century. However, recent evidence suggests that the rate of population decline is enhanced by squirrelpox disease, caused by a viral infection carried asymptomatically by grey squirrels but to which red squirrels are highly susceptible. Population genetic diversity provides some resilience to rapidly evolving or exotic pathogens. There is currently no data on genetic diversity of extant UK squirrel populations with respect to genes involved in disease resistance. Diversity is highest at loci involved in the immune response including genes clustered within the major histocompatibility complex (MHC). Using the class II DRB locus as a marker for diversity across the MHC region we genotyped 110 red squirrels from locations in the UK and continentalEurope. Twenty four Scvu-DRB alleles at two functional loci; Scvu-DRB1 and Scvu- DRB2, were identified. High levels of diversity were identified at both loci in the continental populations. In contrast, no diversity was observed at the Scvu-DRB2 locus in the mainland UK population while a high level of homozygosity was observed at the Scvu-DRB1 locus. The red squirrel population in the UK appears to lack the extensive MHC diversity associated with continental populations, a feature which may have contributed to their rapid decline

Finding Diagnostically Useful Patterns in Quantitative Phenotypic Data.

Trio-based whole-exome sequence (WES) data have established confident genetic diagnoses in ∼40% of previously undiagnosed individuals recruited to the Deciphering Developmental Disorders (DDD) study. Here we aim to use the breadth of phenotypic information recorded in DDD to augment diagnosis and disease variant discovery in probands. Median Euclidean distances (mEuD) were employed as a simple measure of similarity of quantitative phenotypic data within sets of ≥10 individuals with plausibly causative de novo mutations (DNM) in 28 different developmental disorder genes. 13/28 (46.4%) showed significant similarity for growth or developmental milestone metrics, 10/28 (35.7%) showed similarity in HPO term usage, and 12/28 (43%) showed no phenotypic similarity. Pairwise comparisons of individuals with high-impact inherited variants to the 32 individuals with causative DNM in ANKRD11 using only growth z-scores highlighted 5 likely causative inherited variants and two unrecognized DNM resulting in an 18% diagnostic uplift for this gene. Using an independent approach, naive Bayes classification of growth and developmental data produced reasonably discriminative models for the 24 DNM genes with sufficiently complete data. An unsupervised naive Bayes classification of 6,993 probands with WES data and sufficient phenotypic information defined 23 in silico syndromes (ISSs) and was used to test a "phenotype first" approach to the discovery of causative genotypes using WES variants strictly filtered on allele frequency, mutation consequence, and evidence of constraint in humans. This highlighted heterozygous de novo nonsynonymous variants in SPTBN2 as causative in three DDD probands

Prevalence, phenotype and architecture of developmental disorders caused by de novo mutation: The Deciphering Developmental Disorders Study

Individuals with severe, undiagnosed developmental disorders (DDs) are enriched for damaging de novo mutations (DNMs) in developmentally important genes. We exome sequenced 4,293 families with individuals with DDs, and meta-analysed these data with published data on 3,287 individuals with similar disorders. We show that the most significant factors influencing the diagnostic yield of de novo mutations are the sex of the affected individual, the relatedness of their parents and the age of both father and mother. We identified 94 genes enriched for damaging de novo mutation at genome-wide significance (P < 7 × 10−7), including 14 genes for which compelling data for causation was previously lacking. We have characterised the phenotypic diversity among these genetic disorders. We demonstrate that, at current cost differentials, exome sequencing has much greater power than genome sequencing for novel gene discovery in genetically heterogeneous disorders. We estimate that 42% of our cohort carry pathogenic DNMs (single nucleotide variants and indels) in coding sequences, with approximately half operating by a loss-of-function mechanism, and the remainder resulting in altered-function (e.g. activating, dominant negative). We established that most haplo insufficient developmental disorders have already been identified, but that many altered-function disorders remain to be discovered. Extrapolating from the DDD cohort to the general population, we estimate that developmental disorders caused by DNMs have an average birth prevalence of 1 in 213 to 1 in 448 (0.22-0.47% of live births), depending on parental age