bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of hyperattenuated poliovirus as a replacement for Sabin or wild-type strains for laboratory assays in a post eradication world
Authors: Fox Helen , Carlyle Sarah , Minor Philip , Macadam Andrew
Published: April 2019

Poliovirus serotype 2 (PV2) has been declared eradicated and was removed from the live vaccine programme in 2016. WHO have developed the Global Action Plan (GAPIII) to describe containment conditions for laboratory work with poliovirus after eradication, and in the UK, all PV2 has been reclassified to hazard group 3 (HG3). Laboratory work involving live poliovirus will continue to be required years after eradication for the purposes of: vaccine testing; environmental surveillance; immunoglobulin testing; and for the development of new public health interventions. Compliance with GAPIII and HG3 containment conditions would impose financial burden and failure would lead to disruption to essential activities. Development of safer poliovirus strains for use outside of containment could overcome these issues. The S19 poliovirus strain, designed to be hyperattenuated and extremely genetically stable, was used as a cassette for the introduction of capsid proteins of other strains and serotypes (Knowlson et al. 2015), originally for vaccine production. S19 viruses are unable to infect transgenic mice (by intraspinal inoculation) or non-human primates (by mouth) at very high doses and are unlikely to infect humans at biological temperature. PV2 S19 strains have recently been approved for use outside of GAPIII containment requirements by the Containment Advisory Group. Validation of strains for laboratory assays and a highly sensitive QC assay based on NGS will be described.
Role of BPIFA1 in the pathogenesis and immune response against Influenza A virus in mice
Authors: Al Katy Sanaria , Kipar Anja , Stewart James
Published: April 2019

The epithelial layer of the respiratory system has a critical role in the defense against microbes and secretes a number of proteins that function in host defense. BPIFA1 is secreted by the epithelium of the respiratory tract and we have shown previously that it inhibits binding and entry of Influenza A Virus (IAV) into respiratory epithelium (Akram et al. 2018), Mucosal Immunol (11, 71). However, its precise biological functions remain unclear. The aim of this study was to assess the influence of BPIFA1 in antibody production during IAV infection. BPIFA1 KO and wild type C57BL/6J mice were infected with IAV using different virus doses and blood, broncho-alveolar lavage and nasal washes were collected at several time points for analysis of IAV-specific antibodies. The results showed that BPIFA1 has role in the efficient generation of virus-specific IgA in the respiratory tract. Thus, BPIFA1 has an important role not only in innate defense but also in the adaptive immune response against IAV.
PorA-Loop4 derived peptides of Neisseria meningitidis cause a G1 cell cycle arrest through the Akt signalling pathway in human brain microvascular endothelial cells
Authors: Firdaus Rininta , Oldfield Neil J. , Wooldridge Karl G.
Published: April 2019

Neisseria meningitidis (meningococcus) is a major meningitis-causing bacteria and is known for its ability to breach blood-brain barrier (BBB). Meningococcus binds to Laminin receptor (LAMR) on the surface of endothelium, which is part of the BBB. The meningococcal surface proteins PorA and PilQ were previously identified as bacterial ligands responsible for binding and, subsequently, the LAMR-binding moiety of PorA was localised to its fourth extracellular loop (PorA-Loop4). Using a circularised peptide corresponding to PorA-Loop 4 from N. meningitidis MC58, the PorA-LAMR interaction induced specific cellular responses in human brain microvascular endothelial cells (HBMECs) including G1 cell cycle arrest. Flow cytometric analysis indicated that the treatment of HBMECs with PorA-Loop4 for 24 h caused a significant reduction of cells (20 %) at S-phase and a corresponding increase (23 %) in G1 population. Immunoblotting and quantitative real time PCR (qRT-PCR) analysis suggested that a blockade in Akt signalling (key proteins including Akt, GSK-3β, CyclinD1, and CDK4) contributes to the G1 arrest. Immunoblotting showed that the expression of phosphorylated GSK-3β and CDK4 were significantly increased in treated HBMECs. In contrast, the expression of phosphorylated Akt and Cyclin D1 were decreased following treatment. Transcriptome analysis using qRT-PCR confirmed that treatment of HBMECs with PorA-Loop4 peptide for 2, 4, 8, or 24 h increased gene expression of CDK4, and decreased expression of Cyclin D1. Immunofluorescent imaging of Akt, GSK-3β, CyclinD1, and CDK4 in Loop4-treated HBMECs are consistent with qRT PCR and immunoblot results. The data confirm that PorA-Loop4 induce G1 arrest through the Akt signalling pathway via Akt/GSK-3β/CyclinD1/CDK4.
Purification and characterisation of antimicrobial agents isolated from a member of the Paenibacillus genus
Authors: Bennett Vicky , Koch Matthew , Aldarhami Abdu , Upton Mathew
Published: April 2019

Antimicrobial resistance (AMR) poses an ever-increasing threat to public health; the prevalence of resistant bacterial strains has reduced the clinical efficacy of many existing therapeutics and is therefore contributing to rising mortality rates due to difficult to treat bacterial infections. Two key approaches used to mitigate the threat of AMR are the discovery of novel therapeutics with activity against these resistant strains, and educating the wider public about the impact of AMR, and steps that can be taken to reduce the development of resistance. We are combining both approaches to enhance the impact of our public engagement activities. During a recent event at the University of Plymouth, a member of the public isolated the bacterial strain ‘36A’ from the button of a lift control panel. Simultaneous antagonistic screening identified antimicrobial activity against a range of both Gram-positive and Gram-negative bacteria. 36A was then subjected to draft genome sequence determination via the MinION platform (Oxford Nanopore). Growth media were optimised to enhance antimicrobial activity, with fermentation in LB broth and subsequent purification of the culture supernatant via multi-stage column chromatography resulting in the isolation of four putative antimicrobial compounds. Initial characterisation has shown that each compound has a peptidic component, all showing stability and potency at a relatively low concentration against MRSA, E. coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Structural characterisation has been carried out using mass spectrometry, with further characterisation and cell toxicity studies ongoing. The producing strain has been identified as a member of the Paenibacillus genus.
Repurposing histone deacetylase inhibitors (HDACi) to treat Candida glabrata infections
Authors: O’Kane Callum J. , Marshall Andrew , O’Connell Mary J. , Hyland Edel M.
Published: April 2019

Candida glabrata currently accounts for 25 % of all fungal cases in UK hospitals, second only to C. albicans. This number is expected to rise given the intrinsic anti-fungal resistance of this species and the difficulty in treating it. In an effort to identify novel-anti fungal targets in C. glabrata, we used comparative genomics within Saccharomycotina yeast to predict which genes are under positive selection in this species specifically. Such genes are predicted to have influenced the adaptation of C. glabrata from a free-living microbe to a human pathogen, potentially due to functional shift(s) of the proteins they encode. Our analysis predicts that histone acetylation pathways are under positive selection in C. glabrata. Thereforewe hypothesised that we could use histone deacetylase inhibitors (HDACi) to interfere with histone acetylation levels and impact C. glabrata virulence. By treating C. glabrata withbroad spectrum HDACis we show it has a reduced capacity to form biofilms, it is less well adapted to high salt conditions typically found within a human host, and most importantly, it reverts to a more anti-fungal sensitive state. RNAseq analysis indicates that HDACi treatment interferes with the C. glabrata transcriptional response to anti-fungal treatment, rendering it incapable of combating against these drugs. Furthermore, using an in vivo worm model of candidiasis, we show that HDACi treatment in conjunction with the anti-fungal fluconazole, can increase the survival rate of individuals with C. glabrata infections. Taken together our data suggest that the health threat posed by C. glabrata might be addressed by repurposing HDACi to treat this infection.
Novel nitrogen-containing heterocyclics with bactericidal activity against Mycobacterium tuberculosis
Authors: Lanne Alice , Cui Yixin , Salih Mariwan Hama , Craven Philip , Dale Katie , Dodd Helena , Jones Geraint , Lu Xinran , Milne Gavin , Ritchie John , Zhao Yiming , Fossey John , Alderwick Luke
Published: April 2019

Tuberculosis (TB) is the leading cause of global morbidity and mortality caused by an infectious disease, with over 10 million new cases emerging in 2017. This global emergency is exacerbated by the emergence of multi-drug and extensively-drug resistant TB, therefore new drugs and new drug targets are urgently required. From a whole-cell phenotypic screen we identified a series of nitrogen-containing heterocyclic compounds that elicit potent anti-mycobacterial activity with MIC values <10 µM against Mycobacterium tuberculosis. Interestingly, this series of compounds demonstrate no detectable drug resistance in mycobacteria. Mode of action and target deconvolution studies suggest that these compounds inhibit mycobacterial growth by interfering with late-stage mycolic acid biosynthesis. In addition, these compounds exhibit a suitable toxicological and PK/PD profile that paves the way for further development as an anti-TB chemotherapy.
Local genes, for local bacteria: biogeographical variation in Campylobacter accessory genome content
Authors: Pascoe Ben , Sheppard Sam
Published: April 2019

Diarrhoeal disease remains a major cause of child morbidity, growth faltering and mortality in low and middle income countries (LMICs), with Campylobacter among the most common causes. Previous work has identified the major sources in the UK and US (e.g. contaminated poultry), however little is known about the risk factors and transmission routes in LMICs, where incidence is extremely high (up to 85 % of children infected before 1 year) and suggests a different epidemiology. Risk factors such as household crowding, poor sanitation, consumption of contaminated water and cohabitation with animals, all constitute potential transmission risks but their relative importance is unknown. This is a major concern as frequent or chronic enteric (re)infection is linked to significant morbidity and growth faltering in children. Comparative genomics offers a solution for untangling complex transmission networks. Campylobacter jejuni and C. coli primarily inhabit the gut of birds and mammals and signatures of adaptation can be detected in their genomes. Therefore, by sequencing the genome of human clinical isolates and faecally contaminated environments it is possible to discern its origin. Pilot genomics studies of strains from humans, animals and food in LMICs have identified genomic variation in strains that may indicate differences in source, survival, transmission or virulence (compared to the UK). In particular we have identified globally distributed strains and lineages; country-specific strains; rapid dissemination of accessory genes in specific regions; evidence of within-household spread and strains associated with asymptomatic infection or high levels of antimicrobial resistance.
Developing a pseudotyping assay for Zika virus glycoprotein
Authors: Jimenez Fernando Ruiz , Olais Humberto Perez , King Barnabas , McClure Patrick , Ball Jonathan
Published: April 2019

Zika virus (ZIKV) was originally described during 1947 in Uganda when it was isolated from a sentinel macaque in the forest of Zika. After its discovery, discrete outbreaks were reported in Africa and Asia, but it was until 2007 when an outbreak in the Yap Islands where it was identified as a pathogen capable of causing epidemics and became associated with microcephaly. We analysed 10 ZIKV sequences from key locations, tracking the virus’ spread through the Americas, following the Yap Island outbreak. We identified 8 mutations within the M (prM) and E proteins which may alter the tropism or entry kinetics of the virus. To test the influence of these glycoprotein mutants on virus entry we have explored lentivirus pseudotypes with a luciferase reporter to measure infectivity. So far, the model involving the use of PNL 4.3 HIV based retroviral backbone has not given favourable results so in the aim to try to improve the conditions and favour the process we tested different conditions including a matrix of concentrations between the retroviral backbone and the viral glycoprotein. The addition of other viral proteins was also tested in an effort to produce particles capable to infect the target cells.
Wombling surplus diagnostic nucleic acid for novel pathogenesis and genetic epidemiology of viral infections
Authors: McClure Patrick , Clark Gemma , Chellapuri Akhil , Smitheman Matthew , Bagasi Arwa , Akagha Terry , Khandaker Tasneem , Howson-Wells Hannah , Chappell Joseph , Tsoleridis Theocharis , Ball Jonathan , Irving William
Published: April 2019

Nottingham University Hospitals Trust receives circa 13 000 samples for diagnosis of respiratory and neurological viruses per annum, however positive results are achieved in approximately 50 % of respiratory and 20 % of neurological investigations. We therefore aimed to retrospectively extend the diagnostic spectrum for these samples by applying a battery of degenerate PCR assays to surplus diagnostic nucleic acids.218 previously negative respiratory specimens collected in 2016 from children under 5 years old, identified positivity of 11 % for Human Bocavirus, 5 % for Human Enteroviruses (including 3 cases of Enterovirus D68), 4 % for Human Coronaviruses and one individual positive for Trichodysplasia Spinulosa Polyomavirus. Complementary investigation of 1730 previously negative specimens from children and adults with neurological symptoms yielded positive results for Hepatitis E, BK Polyomavirus and Astroviruses in addition to Entero- and Parechoviruses apparently missed by standard diagnostic assays. Our extensive archive further allowed us to investigate relatively rare viral infections in significant numbers. Therefore we also studied the genetic and clinical epidemiology of the human Rubulavirus pathogens Parainfluenza 2 and 4 in 121 and 237 patients respectively between 2013 and 2017. This indicated co-circulation of three clusters of Parainfluenza 4 in Nottingham with greater presentation of subtype 4b than 4a and 2 clades of Parainfluenza 2. 5 fatalities were recorded in the Parainfluenza 2 cohort. In summary, surplus nucleic acid from viral diagnostic laboratories represents a valuable resource for both service development and clinical research. Coronavirus, Bocavirus and Enterovirus testing have since been implemented in the routine diagnostic panel for respiratory investigations.
Industrially useful microbial species for production of biofuels and chemicals from municipal solid waste
Authors: Dornau Aritha , Thomas Gavin , McQueen-Mason Simon
Published: April 2019

Municipal solid waste (MSW) production is projected to reach 3.4 billion tonnes per annum by 2050. The majority of MSW produced globally is incinerated or diverted to landfill, both methods which pollute the environment and contribute substantially to climate change. The organic fraction of MSW (OMSW) typically comprises ∼50 % lignocellulosic material and presents an abundant renewable feedstock for producing biofuels and chemicals. An important step toward OMSW valorisation is the identification of suitable microorganisms capable of fermenting this highly inconsistent, heterogeneous and complex feedstock. We have characterised the fermentation performance of eight biotechnologically relevant microorganisms (Clostridium saccharoperbutylacetonicum, Escherichia coli, Geobacillus thermoglucosidasius, Pseudomonas putida, Rhodococcus opacus, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Zymomonas mobilis) on enzymatic hydrolysate of OMSW fibre produced by a commercial autoclave pre-treatment. S. cerevisiaewas the most efficient ethanol producer, followed closely by Z. mobilis. Both species produced ethanol to high titre within 24 h, but neither could ferment xylose. The most effective performance was demonstrated by R. opacus, which consumed all available glucose and xylose concurrently over 72 h and produced a remarkably large yield of triacylglycerol (a biodiesel and aviation fuel precursor). This work demonstrates that OMSW is a promising renewable feedstock capable of supporting the growth several industrially useful microorganisms to high product titres. The best performing species identified here are interesting candidates to study further for application in a MSW biorefinery.
Coinfection promotes plasmid stability
Authors: Aguado Laura Carrilero , Hall James P. J. , Brockhurst Michael
Published: April 2019

Bacteria exist in complex communities that include not only many other bacterial species but also diverse mobile genetic elements which accelerate bacterial evolution via horizontal gene transfer. How multiple mobile genetic elements interact in bacterial populations and how this affects plasmid dynamics remains poorly understood. We experimentally evolved populations of Pseudomonas fluorescens SBW25 either singly-infected or co-infected with two conjugative mercury resistance plasmids either with or without positive selection (i.e. addition of Hg(II)). We show that co-infection led to higher levels of mercury resistance in the bacterial population in the absence of positive selection. Consistent with this, in the absence of positive selection the plasmids could stably coexist within bacterial cells resulting in the maintenance co-infection. By contrast, with positive selection, plasmid coexistence was destabilised, leading to the dominance of a single plasmid in several replicate bacterial populations. Plasmid co-infection appears to alter the trajectory of compensatory evolution to ameliorate the cost of plasmid carriage and may have selected for alternative mechanisms compared to singly-infected populations. Stable plasmid co-infection without positive selection for plasmid-encoded traits suggests that environments where plasmids are useless may be hot-spots for genomic innovation via plasmid-plasmid recombination.
A longitudinal survey of the gut virome of commercial broiler chickens from prehatch to slaughter
Authors: Trudgett James , Brennan David , Casement Veronica , McAdam Paul , Smyth Victoria
Published: April 2019

Uneven flock growth (UFG) occurs when a broiler flock exhibits a wide range of bodyweights at slaughter. Automated chicken processing plants operate on an average bodyweight for the flock being processed so UFG can cause disruption to the processing flow that requires manual remediation. It is known that enteric virus infections of young broiler flocks lead to varying degrees of growth restriction that range from runts at hatch and severe early stunting, which are generally culled, to poor flock performance and UFG. In order to investigate the make-up of communities of enteric viruses (virome) associated with poor growth and also their infection timings and persistence in broiler flocks, a longitudinal survey of 7 commercial broiler flocks of varying performance over 3 successive broiler crops was undertaken with gut and faecal samples collected from 50 birds at each of 12 timepoints from pre-hatch to slaughter. The samples were pooled for each timepoint, then processed to enrich for viruses by removing host cells and bacteria. The RNA was extracted from each timepoint sample, amplified by whole transcriptome amplification followed by whole genome library preparation for the Illumina MiSeq platform and libraries multiplexed. Bioinformatics analysis used ViromeScan which facilitates metagenomic studies of quality-trimmed sequencing reads. The presence of diverse viral families was seen including the known major enteric viruses of the astrovirus, picornavirus, reovirus and parvovirus families. A greater diversity of viruses was observed in the flocks of poorer performance than in those flocks of good performance.
Human immunodeficiency virus type 2 (HIV-2) dynamics and whole genome deep sequence analysis in Mauritian-origin cynomolgus macaques (Macaca fascicularis)
Authors: Ham Claire , Ferguson Debbie , Jenkins Adrian , Hall Jo , Almond Neil , Berry Neil
Published: April 2019

Human immunodeficiency virus type 2 (HIV-2) is a pathogenic human retrovirus with a distinct natural history and lineage derivation from pandemic HIV-1. HIV-2 infections in humans are the result of zoonotic transmission from sooty mangabey monkeys naturally infected with SIVsm. Unlike HIV-1, HIV-2 infects other non-human primate species, including baboons and macaques. We determined the infectivity and infection kinetics of a Gambian-origin HIV-2 isolate HIV-2SBL6669 strain in Mauritian-derived cynomolgus macaques (MCM) in the context of a heterologous superinfection resistance study. The ability of HIV-2 to replicate in unvaccinated MCM with limited host genetic MHC and TRIM5 spectrum was determined where the kinetics of plasma HIV-2 RNA mimic the phenotypic response typically observed in HIV-2-infected humans. HIV-2SBL669 replication is not completely restricted in this species and may establish a persistent infection. This is determined by moderate peak viral loads (105–6 log10 HIV-2 RNA copies/ml) controlling to a low level determined by qRT-PCR and detectable in-situ hybridisation signals in lymphoid tissue 20 weeks after challenge. In MCM vaccinated for 20 weeks with an attenuated SIV, we observed high levels of superinfection resistance as determined by virus-specific PCR analysis of tissues and low total plasma viral RNA levels in vaccinates compared to unvaccinated HIV-2 controls. Recovery of whole viral genome and next generation sequence analysis of challenge controls, including low-level breakthrough variants characterises viral challenge and vaccine-escape viruses. HIV-2 in cynomolgus macaques recapitulates many of the features of HIV-2 infections in humans.
Pigs, PAMs and Pathogens: using primary alveolar macrophages from abattoir acquired porcine lungs to model pulmonary infections
Authors: McNicholl Johanna , Maurizio Illaria , Yugueros David Carrenos , Andrew Peter , Oggioni Marco
Published: April 2019

The similarity between the porcine and human immune system makes pigs an ideal model for studying infectious disease. We have been using alveolar macrophages recovered from abattoir-sourced porcine lungs to create a model to study Streptococcus pneumoniae infections in the lung. Pneumonia is a leading cause of death from infectious disease, with S. pneumoniae the most common cause of bacterial pneumonia. Although much studied, there is still a lot we don’t know about the early stages of infection. Recent research in our lab has found a previously unknown intracellular phase for S. pneumoniae within splenic macrophages Alveolar macrophages were recovered by bronco-alveolar lavage and used in gentamicin protection assays. Macrophages challenged with S. pneumoniae at an MOI of 25 bacteria per cell were found to contain live bacteria up to 5 h after infection. Analysis of samples collected at 45, 90, 180 and 300 min after challenge found less than 10 % of the challenge dose present at 45 min, decreasing to 0.02 % after 5 h. The presence of bacteria inside the macrophages was confirmed by confocal microscopy. Studies are on-going to unpick the processes that allow the bacteria to resist phagocytosis by the macrophages, and to use the model to investigate the interaction of the macrophages with other respiratory pathogens. The use of abattoir-acquired porcine cells contributes to the 3Rs – reducing and replacing laboratory animals, and refinement of method as pigs are more akin to humans than mice. J. McNicholl is sponsored by the Daphne Jackson Trust.
Using human iPSC derived small intestinal organoids as a model for enteric disease caused by Enterotoxigenic E. coli and Vibrio cholerae
Authors: Kane Leanne , Hale Christine , Goulding David , Dorman Matthew J , Clare Simon , Wren Brendan , Thomson Nicholas
Published: April 2019

Within the last ten years, iPSC (induced pluripotent stem cells) have been widely shown to have the ability to be re-programmed to produce a wide range of tissues in the presence of certain growth factors. In this project, we re-direct human stem cells derived from fibroblasts into complex 3D small intestinal structures termed organoids. These organoids have been shown to possess all cell types that are present in small intestinal tissue such as, enterocytes, goblet cells, enteroendocrine cells and Paneth cells, as well as possessing microvilli and crypt structures. We demonstrate that it is possible to microinject into the lumen of these small intestinal organoids and to manipulate the conditions for infection of non-invasive bacteria such as Enterotoxigenic E. coli (ETEC) and Vibrio cholerae. Looking at known bacterial virulence factors, we have shown there are differences in patterns of infection among different strains of entero-pathogenic bacteria. In addition, we have shown that the induced human organoids (iHO) elicit a recognisable and measurable host response to bacterial toxin.
Investigating the pangenomes of microbial eukaryotes
Authors: McCarthy Charley , Fitzpatrick David
Published: April 2019

Pangenomes evolve in prokaryotes as a result of promiscuous horizontal transfer of genetic material and other rapid evolutionary processes. Although eukaryotes are generally under more restrictive evolutionary constraints than prokaryotes (e.g. lower levels of HGT), pangenomic structure has also been identified in plant, algal and fungal species. A number of methodologies for pangenome analysis have been published in recent years, but these methodologies are generally specifically-intended or optimized for prokaryotes. We have developed a software pipeline based around the previously-published PanOCT method of pangenome construction with additional pre- and post-processing methodologies tailored for eukaryote pangenome analysis. We have previously used this pipeline to analyze the pangenomes of model fungal species such as Saccharomyces cerevisiae, and we are currently testing our pipeline by constructing and analyzing the pangenome of the industrially-relevant yeast species Yarrowia lipolytica. Analysis of fungal pangenomes shows that core and accessory eukaryotic species genomes encompass a variety of phenotypes and suggest that gene duplication events play a larger role in eukaryote pangenome evolution than HGT.
Studying a mycovirus from Dothistroma septosporum, causative agent of pine needle blight
Authors: Daudu John , Snowden James , Tubby Katherine , Coutts Robert , Kotta-Loizou Ioly
Published: April 2019

Dothistroma needle blight caused by D. septosporum has emerged in the British Isles as a major threat to Corsican pine, lodgepole pine and Scots pine. There is increasing evidence that mycoviruses can reduce the growth and pathogenicity of fungal plant pathogens. The aim of the present study is to characterise a double-stranded RNA virus found in D. septosporum and investigatefor putative hypovirulence, a common feature noted for mycoviruses, which might be used for biological control to invasion by more aggressive strains of the fungus. To this endthe viral genome was cloned and sequenced revealing four genomic segments, each one containing a single open reading frame (ORF) flanked by 5’ and 3’ untranslated regions. The ORFs encode the RNA-dependent RNA polymerase, the capsid protein, a protein of unknown function and a putative protease, respectively. Phylogenetic analysis of the sequences obtained revealed their similarity to members of the established family Chrysoviridae, genus Alphachrysovirus, which are encapsidated in isometric particles and are known to elicit hypovirulence in their hosts. Subsequently, virus-free and virus-infected isogenic lines were generated to determine any effects of the mycovirus on fungal fitness and pathogenicity. More specifically, the virus-infected isolate is currently being assessed in comparison to the virus-free one in terms of radial growth in solid culture, biomass in liquid culture, pathogenicity in pine trees and production of the mycotoxin dothistromin. In conclusion, this study reports the first mycovirus ever found in D. septosporum.
Biomineralization of microbes in natural and restored saltmarshes: a missing link in restoration efforts?
Authors: D’Arcy James , Antunes André
Published: April 2019

Saltmarshes are threatened coastal ecosystems, which are subject to cyclic variation in conditions which create a gradient-rich environment (e.g. salinity, pH). They support very diverse communities of plants and animals, protect the coast from erosion, and play an important role in element cycling and bio-remediation. The importance of saltmarshes has only been recently recognised, which has led to significant efforts in restoring sites that had been previously converted to Agriculture. Such restoration attempts have had limited success in returning these sites to their original biodiversity and biological structure. While not yet fully understood, it is thought that several factors are at play, including persisting differences in soil structure and quality. Coastal environments have been previously shown to harbour significant microbial populations capable of producing CaCO3 biominerals. On the other side, CaCO3 biomineral production is known to affect the texture and overall properties of soil, a property currently used in the improvement of soils for Agriculture. We suggest that these factors could be linked, and that the limitations of saltmarsh restoration efforts might result from differences in biomineral production. Our results provide an overview on differences between samples that we’ve collected in natural and restored sites, based on cultivation and screening efforts. Our insights might provide an important step forward in saltmarsh restoration and contribute to more successful approaches to protect these vital biotopes and increase biodiversity.
Do Streptococcus pneumoniae and respiratory Syncytial virus synergise to promote invasive disease?
Authors: Shadia Khandaker , Yang Marie , Kadioglu Aras
Published: April 2019

Streptococcus pneumoniae (S.p.) and Respiratory Syncytial Virus (RSV) are two major pathogens commonly found to coexist in respiratory secretions in patients with acute respiratory infection. Though there is increasing evidence of a synergistic interplay between these two pathobionts, the exact mechanisms remain obscure. The aim of our study was to decipher how coinfection with RSV alter pneumococcal growth dynamics and host immune response and how this impact on the colonisation and invasive properties of S.p. Using in vivo mouse model, we made the key observation that upon coinfection with RSV, the density of pneumococcal colonisation in the nasopharynx and dissemination to the lower respiratory tract were significantly higher in mice previously colonised with S.p. These mice also presented more severe weight loss and delayed recovery compared to mono-infected animals as well as significantly heightened pro-inflammatory cytokine profiles. Measurement of in vitro transepithelial electrical resistance (TEER) showed that, upon RSV coinfection, S.p. transmigrate through the epithelial barrier without altering epithelial integrity suggesting a transcellular mechanism rather than paracellular migration. Moreover, RSV-pneumococcal coinfection of human primary nasal epithelial cell demonstrated major changes in host protein expression involved in the catalytic activity, ubiquitination, cytoskeletal organisation, and endocytosis. Simultaneously, significant upregulation observed in bacterial proteins involved in the ribosomal activity, streptococcus-induced tissue inflammation, DNA supercoiling, and bacterial viability during oxidative stress, affecting both the survival and the virulence of S.p. Our results explain the complex interactions between pneumococci, RSV and host and help towards further understanding the significance of viral-bacterial co-infection in clinical settings.
Development of ‘smart’ wound dressings for biofilm sensing and control
Authors: Magee Erin , Wells Nathan , Mills Andrew , Gilmore Brendan
Published: April 2019

Chronic wounds affect approximately 2 % of the worldwide population and incur healthcare costs in the billions. Key to their persistence is the formation of microbial biofilms, which are accounted for in nearly 80 % of all non-healing wounds. The smart dressing presented herein aims to detect a range of volatile infection protagonists, with a striking colour change that can be visualised with the naked eye, providing 24/7, non-invasive monitoring of infection development and antimicrobial treatment efficacy. A range of coloured indicator films housing dyes responsive to volatile analytes in the wound headspace were developed and tested against porcine skin inoculated with Pseudomonas aeruginosa. Digital images of the indicator film were captured at regular time intervals and the resulting images were aligned and split into red, green and blue (RGB) colour channels to yield semi-quantitative data. vAPCI-MS was exploited to identify additional volatiles for incorporation into the smart dressing design. A CO2-sensing film comprising xylenol blue dye underwent a marked colour change from blue to yellow within 12 h of inoculation with PAO1, whilst indicators monitoring uninoculated control skin remained blue (no colour change). In addition, vAPCI-MS identified putrescine as an additional volatile of interest, and responsive indicator films were developed for its detection. The marked colour change exhibited by each indicator film is easily visualised by eye and can be digitally analysed to provide semi-quantitative data. This early warning, point-of-care technology is a promising candidate in combatting biofilm development in wounds.
Characterising the insect-borne transmission of the cattle poxvirus lumpy skin disease virus
Authors: Sanz-Bernardo Beatriz , Wijesiriwardana Najith , Haga Ismar , Gubbins Simon , Hawes Pippa , Wilson Anthony , Sanders Christopher , Basu Sanjay , Alphey Luke , Langlands Zoe , Darpel Karin , Beard Pip
Published: April 2019

Lumpy skin disease (LSD) is an emerging poxviral disease of cattle caused by the Capripoxvirus lumpy skin disease virus (LSDV) which generates widespread cutaneous lesions in affected animals. LSD is recognised as a transboundary high consequence disease in Africa where it contributes to rural poverty and food insecurity. In 2015 LSDV spread to southeastern Europe and currently poses a threat to cattle in neighbouring regions. Previous research indicates that LSDV is most likely transmitted by insect vectors however details of transmission pathways are unclear. This study was designed to identify the risk of transmission of LSDV posed by different insect vectors. A bovine experimental model of LSD was established in the high containment facilities at The Pirbright Institute. Potential insect vectors (Aedes aegypti, Culex quinquefasciatus, Stomoxys calcitrans and Culicoides nubeculosus) were fed on LSDV-infected cattle then incubated for up to eight days. Cattle and insects were regularly sampled to quantify LSDV present in different tissues and vector species. This data was then used to model the dynamics of LSDV infection and transmission. All four species of vector successfully acquired LSDV from infected cattle and maintained the virus up to eight days post feeding. The outputs of this research will now be used to design more effective LSD control programmes.
The utilisation of organoids and macrophages derived from Human induced pluripotent stem cells as model systems to investigate host-bacterial interactions
Authors: Hale Christine , Kane Leanne , Dorman Matthew , Thomson Nicholas
Published: April 2019

Using human induced pluripotent stem cell (hiPSC) technology we are developing methods to examine host-bacterial interactions. Due to the fact that undifferentiated human induced pluripotent stem cells are amenable to genetic engineering, can be cultured indefinitely and can further be differentiated into multiple cell types, we are exploiting both organoid and macrophage systems to investigate the interactions between host cells and diarrhoeal pathogens, including enterotoxigenic Escherichia coli and Vibrio cholerae. Utilising both wild type and relevant knockout hiPSC lines we are probing both initial interactions and subsequent utilisation of pathways for the effects of toxins. The further analysis of genetically engineered bacteria extend the usefulness of this model system, and complement the availability of mutant host cells, towards the simultaneous genetic analysis of both pathogen and host.
Proteomic analysis of three ubiquitous phytophthora species threatening global forest ecosystems
Authors: McGowan Jamie , O’Connor Eoin , O’Hanlon Richard , Owens Rebecca A , Fitzpatrick David A
Published: April 2019

Phytophthora are a genus of microbial, filamentous eukaryotes that morphologically resemble fungi but belong to the Oomycete class. Phytophthora species include some of the most destructive pathogens of plants, including many economically important crops and forest species. They represent one of the biggest threats to worldwide food security and natural ecosystems. Phytophthora are notorious for secreting large arsenals of effector proteins which facilitate infection by degrading host cell components, exploiting host nutrients, dampening host immune responses and inducing necrosis. Compared to other taxonomic groups, there is a paucity of OMICs data available to study Phytophthora species. To this end, we have used an LC-MS/MS strategy to perform the first large-scale profiling of the secretomes of three Phytophthora species that are an increasing threat to global forest ecosystems: Ph. chlamydospora, Ph. gonapodyides and Ph. pseudosyringae. Together, Ph. gonapodyides and Ph. chlamydospor are present the two most widespread Phytophthora species, having been found in a wide range of habitats globally. Ph. pseudosyringae has been identified as the cause of oak and beech decline across Europe and America. Here, we use mass spectrometry to characterise the secretome of these Phytophthora species by identifying proteins secreted into different growth media. We detect a number of important effector families including proteins involved in the breakdown of plant cell wall carbohydrates (CAZymes) and toxin families such as necrosis-inducing proteins. Our results provide important insights into understanding the molecular mechanisms of Phytophthora infection.
Induction of inflammasome-dependent signalling in the human monocytic cell line THP-1 by Campylobacter lipooligosaccharides
Authors: Hameed Amber , Machado Lee , Woodacre Alexandra , Marsden Gemma
Published: April 2019

The Campylobacter lipooligosaccharides (LOS) can stimulate membrane-bound innate immune receptors in human macrophages. However, the association of Campylobacter LOS in the stimulation of cytosolic receptors or the inflammasome remains poorly characterised. Therefore, the aim of this study was to determine the role of Campylobacter LOS in the activation of NLRP3 inflammasome-dependent signalling in a human monocytic cell line. The induction of NLRP3 inflammasome-mediated IL-1β and Caspase-1 secretion in THP-1 supernatants was quantified using ELISA following co-culture of THP-1 cells with LOS extracts from wild type C. jejuni 11168, mutant C. jejuni 11 168 with reduced LOS and two wild type C. coli strains (RM1875 and 76339). Our results demonstrate that LOS purified from both C. jejuni and C. coli can induce Caspase-1 and IL-1β production in human macrophages. However, C. jejuni 11 168 mutant LOS with modified lipid A and lack of core oligosaccharides stimulated significantly reduced Caspase-1 and IL-1β. This result was also replicated in co-culture of live wild type and mutant C. jejuni with THP-1 cells. This study provides new insight into the interaction of Campylobacter with human macrophages and suggests that variation in LOS structure may alter NLRP3 inflammasome activation.
HIV-1 infectivity of cells is enhanced at mitosis: a role for Vpr?
Authors: Gibbons Joseph , Lee Wing-yiu Jason , McKnight Áine
Published: April 2019

Restriction factors are present in all cells including non-immune cells and protect them from invading viruses. RNA-associated Early-stage Antiviral Factor (REAF) was identified from a whole genome siRNA screen for restriction factors to HIV-1(1). siRNA induced knockdown of REAF increases viral infectivity, while over-expression of the protein limits infection(2). Here we show that during mitosis, REAF is specifically excluded from the chromatin region and that mitotic cells have an increased susceptibility to HIV-1. We also demonstrate that in HIV-1 infection of monocyte-derived macrophages, Vpr is responsible for the degradation of nuclear REAF. Furthermore, silencing REAF expression in cycling cells by RNAi causes cells to accumulate in the G2/M phase. This result is consistent with previous observations that Vpr induces cell cycle arrest after infection (3).

1. L. Liu et al., A whole genome screen for HIV restriction factors. Retrovirology 8, 94 (2011).

2. K. M. Marno et al., Novel restriction factor RNA-associated early-stage anti-viral factor (REAF) inhibits human and simian immunodeficiency viruses. Retrovirology 11, 3 (2014).

3. J. B. Jowett et al., The human immunodeficiency virus type 1 vpr gene arrests infected T cells in the G2+M phase of the cell cycle. J Virol 69, 6304–6313 (1995).
Investigating methane mitigation in beef cattle fed with natural additives
Authors: Ornaghi Mariana , do Prado Rodolpho , Oyama Linda , Huws Sharon , do Prado Ivanor
Published: April 2019

Antibiotics are extensively fed to beef cattle as they act as rumen modulators, improving animal efficiency and decreasing methane emissions. However, current recommendations by health agencies to limit/ban antibiotic use in animal production call for alternatives such as natural additives. In this study, we aimed to evaluate the efficacy of natural additives in methane mitigation. Bulls (½Angus and ½Nellore) 16±2.2 months old, with average body weight of 385±20.7 kg were fed a basal diet (70 % concentrate, 30 % corn silage) offered ad libitum for 62 days in a feedlot and randomised on five treatments (8 bulls/treatment): control treatment, and addition of 1.5, 3.0, 4.5, or 6.0 g/day/animal of a blend of natural additives containing 37.5 % each of clove essential oil, the commercial blend containing vanillin, eugenol and thymol, 12.5 % and 12.5 % of castor and cashew oils). Methane production from rumen fluid was estimated based on the theoretical fermentation balance for observed molar distribution of VFAs in the rumen. DNA extracted from rumen fluid were sequenced and analysed for methane genes within the MG-RAST database. The natural additives linearly reduced methane production (76 %, P<0.02). Evaluation of Archaeaabundance showed a reduction (79 %, P<0.05) in the major methane producing genera: Halorhabdus, Ferroplasma, Methanoplanus, Picrophilus. A reduction of Fibrobacter and Lactobacillus (71 %), the greatest producers of acetate releasing hydrogen for methane formation was also observed. Our findings suggest that natural additives such as essential oils may be useful in the mitigation of greenhouse gases such as methane in animal production.
Dairy processing wastewater as a feedstock for microbial bioplastic production
Authors: Fricke Arno , Murphy Jerry D. , O’Leary Niall D.
Published: April 2019

The end of the European milk quotas in 2015 resulted in a steep increase of Irish milk production from 5 to over 7.2 billion litres annually. Dairy processing ads value, but generates up to 10 litres wastewater (WW) per litre processed. Organic pollutants in the WW need to be removed before discharge. Our approach aims to turn this waste into a resource for polyhydroxyalkanoates (PHA), bioplastic production. Bioplastics are promising materials to reduce our dependency on fossil fuels and waste production. The first production step includes an adaption of the dairy processing WW in an anaerobic, hydrolytic reactor, where acidogenic bacteria metabolise the organic fraction of the WW and form volatile fatty acids (VFAs) the building blocks of PHA. The second step comprises the adaption of biomass towards PHA accumulation. The adaption is driven by an aerobic dynamic feeding strategy to increase the formation of the storage molecule PHA. Addition of the WW as substrate is followed by a starvation period, where bacteria capable of PHA storage have a selection advantage. Over time the mixed microbial system is therefore optimised for PHA accumulation. In a final production step the adapted WW and biomass is combined in a fed-batch reactor to produce the end product PHA. The laboratory scale system could be established and is currently optimised. A later scale-up will help to assess the full economic potential of this waste to value approach.
Replicative fitness and transmission of G57 lineage and UDL01 like H9N2 viruses in chickens
Authors: Bhat Sushant , Shelton Holly , Sadeyen Jean-Remy , Iqbal Munir
Published: April 2019

The low pathogenic H9N2 influenza viruses are a threat to poultry as well as global public health due to their ability to reassort with other avian influenza viruses leading to the emergence of novel reassortant viruses having pandemic potential. The continued inter-subtypic reassortment events between influenza viruses in the Indian sub-continent have led to the replacement of the already existing G1 lineage of H9N2 viruses with the UDL genotype-like (A/chicken/Pakistan UDL-01/08/H9N2) viruses, which are triple reassortants between H9N2 virus (G1 lineage), HPAI H5N1 virus (clade 2.2) and HPAI H7N3 viruses. G1 lineage of H9N2 viruses in China has also been replaced with a fitter G57 lineage which donated internal genes to novel H7N9 viruses in 2013. We assessed and compared the replication, transmission and pathogenic potential of UDL01/2008/H9N2 virus and A/Ck/Vietnam/H7F-14-BN4-315/2015 H9N2 virus of G57 lineage isolated from Vietnam in 2015. Vietnam H9N2 virus was found to be relatively more virulent compared to the UDL genotype-like H9N2 in Chickens. Our in-vitro and in-ovo infection studies also showed that Vietnam/BN4-315/H9N2 virus has greater replication fitness compared to UDL-01/08/H9N2 virus. The UDL-01/08 H9N2 reassortants carrying internal genes of Vietnam/BN4-315 virus also showed improved replication fitness in MDCK cells. It is, therefore predicted that genetic reassortment between dominant strains in the Far East and the Indian subcontinent/Middle East may generate more virulent H9N2 viruses.