bluuebunny/crossref_metadata_2025 huggingface dataset:
Aberrant RNA replication products of highly pathogenic avian influenza viruses and its impact in the mammalian associated cytokine storm
Authors: Penn Rebecca , David-Sanchez Raul-Yusef , Long Jason , Barclay Wendy
Published: April 2019

Highly pathogenic avian influenza viruses (HPAIVs), can sporadically cross the species barrier and cause zoonotic infections in mammalian hosts (including humans), with often fatal consequences. Severe disease has been associated with an overexuberant host innate immune response known as hypercytokinema (cytokine storm) where excessive levels of pro-inflammatory cytokines are produced. Previous work in our laboratory shows that high levels of viral replication by HPAIV in murine myeloid immune cells correlated with high cytokine levels and mapped this phenotype to the polymerase genes. Innate sensing of IAV is performed by the cytoplasmic helicase RIG-I, which recognises blunt ended double stranded RNA with 5’-triphosphate extremities, a pattern present in the viral genome. Defective viral genomes (DVGs) can be produced by aberrant RNA replication and these are also recognised by RIG-I and could play a role in hypercytokinemia. Our studies aim to probe for the presence of DVGs in influenza infected cells in vitro, and in lung samples from infected mice in vivo as well as in murine immune cells ex vivo, using RT-PCR and sequencing. We will establish whether DVGs are correlated to pathogenicity and high pro-inflammatory cytokine levels. Preliminary data utilising minigenome assays show that the polymerase genes from an HPAIV H5N1 strain do generate DVGs, whereas DVGs could not be detected from a H3N2 seasonal polymerase. Ultimately, we aim to identify mutations within the polymerase genes that contribute to virulence and by using reverse genetics, create mutant viruses that test the hypothesis that aberrant polymerase activity drives hypercytokinemia.
The secretome profiling of a pediatric airway epithelium infected with human respiratory syncytial virus (hRSV) identified aberrant apical/basolateral trafficking and novel immune modulating (CXCL6, CXCL16, CSF3) and antiviral (CEACAM1) proteins
Authors: Touzelet Olivier , Broadbent Lindsay , Armstrong Stuart , Aljabr Waleed , Cloutman-Green Elaine , Power Ultan , Hiscox Julian
Published: April 2019

Rationale

The respiratory epithelium comprises polarized cells at the interface between the environment and airway tissues. Polarized apical and basolateral protein secretions are a feature of airway epithelium homeostasis. Human respiratory syncytial virus (hRSV) is a major human pathogen that primarily targets the respiratory epithelium. However, the consequences of hRSV infection on epithelium secretome polarity and content remains poorly understood.

Objective

To investigate the impact of hRSV on the secretome of pediatric respiratory epithelium.

Methods

A proteomics approach was combined with an ex-vivo pediatric airway epithelial model (HAE) (n=3 donors) of hRSV infection to identify the apical and basolateral secretome of hRSV-infected cultures.

Measurements and main results

Following hRSV infection, many host proteins lost their apical- or basolateral-restricted secretion or displayed altered apical/basolateral abundance ratios. Fifty three proteins were specifically associated with RSV infection, including modulators of neutrophil and lymphocyte activation (CXCL6, CSF3, SECTM1 or CXCL16), and antiviral proteins (BST2 or CEACAM1) that were never previously associated with hRSV. Importantly, CXCL6, CXCL16, CSF3 was also detected in nasopharyngeal aspirates (NPA) from hRSV-infected infants but not non-infected controls. Furthermore, the antiviral activity of CEACAM1 against RSV was confirmed in vitro using BEAS-2B cells.

Conclusions

hRSV infection disrupted the polarity of the pediatric respiratory epithelial secretome. It also specifically induced immune modulating (CXCL6, CXCL16, CSF3) and an antiviral protein (CEACAM1) that are new to hRSV infection or disease. This study, therefore, provides novel insights into RSV pathogenesis and endogenous antiviral responses in pediatric airway epithelium.
Acquisition of fluoroquinolone resistance in Campylobacter jejuni leads to an increase in biofilm formation and virulence
Authors: Whelan Matthew , Simpson Jeremy , Cróinín Tadhg Ó
Published: April 2019

Campylobacter jejuni is the leading cause of bacterial gastroenteritis with over 550 million cases reported yearly. The World Health Organization has listed C. jejuni as one of 12 microorganisms on a global priority list for antibiotic resistance due to a rapid increase in the number of strains resistant to fluoroquinolone antibiotics. This fluoroquinolone resistance is conferred through a single point mutation in the QRDR region within the gyrA gene which is also involved in DNA supercoiling homeostasis. We recently revealed that changes in DNA topology play a major role in the regulation of virulence in C. jejuni with relaxation of DNA supercoiling associated with increased attachment to and invasion of human epithelial cells. The aim of this study was to investigate whether fluoroquinolone resistant strains of C. jejuni displayed altered supercoiling associated phenotypes. A panel of mutants were derived against nalidixic acid and ciprofloxacin and shown to have a greater ability to form viable biofilms under aerobic conditions and that this phenotype was associated with changes in DNA supercoiling levels. These mutants were also shown to have an increased ability to attach to and invade epithelial cells in vitro and conferred an increase in the killing efficiency of Galleria mellonella. We report for the first time that fluoroquinolone resistance in C. jejuni is associated with an increase in virulence and the ability to form viable biofilms in oxygen rich environments. These altered phenotypes may play a critical role in the continued increase in fluoroquinolone resistance observed for this important pathogen.
A spatial proteomic approach to identify members involved in the IFN response to HCMV
Authors: Davies Colin , Potts Martin , Itzhak Dan , Nightingale Katie , Davies Alex , Antrobus Robin , Williamson James , Borner Georg , Weekes Mike
Published: April 2019

During the early stages of viral detection, signalling and translocation by key protein mediators initiate antiviral activity. For example, interferon (IFN) signals through the JAK/STAT pathway, leading to phosphorylation of STAT1/2 and movement in complex with IRF9 into the nucleus. To identify novel proteins involved in the IFN response to viruses, we have employed a variety of unbiased, spatial proteomic screens that detect translocation of proteins between different organelles on a global scale. Using three different spatial screens in IFN-stimulated or control human foreskin fibroblasts, we quantified movement of >8000 proteins between the nucleus, cytoplasm and individual organelles. A novel method enabled simultaneous analysis of >2000 phosphoproteins. Proteins were shortlisted based on significant, reproducible movement. Results were validated by appropriate movement of STAT and IRF9 controls. To focus on proteins involved in the IFN response that are important during HCMV replication, we used a complementary screen of proteins that are rapidly downregulated or degraded by HCMV. The results of these screens will be presented, including a shortlist of novel factors that may have new roles in the IFN response to HCMV.
From trash to treasure – turning plastic waste into biodegradable polymers using bacteria
Authors: Johnston Brian , Kowalczuk Marek , Hill David , Tchuenbou-Magaia Fideline , Jonah Itohowo , Radecka Iza
Published: April 2019

Mountains of plastic waste consisting of carrier bags and medical packaging are buried in landfill sites dumped in rivers around the world annually. Unfortunately plastics generated by the petrochemical industry are not biodegradable and therefore accumulate in the environment at a rate of over 25 million tones year-1. Therefore there is a huge demand for biodegradable plastics. Polystyrene (PS), polyethylene (PE) and polypropylene (PP) are problematic materials, used for appliance housings, disposable cutlery and general packaging. This study investigates the utilisation of waste PS, PE and PP as a potential additional carbon sources using bacteria to synthesise polyhydroxyalkanoates (PHAs); a value-added material, able to replace some conventional fossil-fuel plastics while being non-toxic, fully biodegradable and biocompatible. Prodegraded waste PS and PP, and thermally treated PEwere used as supplementary carbon sources totryptone soya broth (TSB and BSM) for 48 h fermentations [1, 2, 3]. The bacterial strain Cupriavidus necator H16 was selected as it is non-pathogenic, genetically stable, robust and one of the best natural producers of PHA. The accumulation of PHAs varied from 17 % (wt / wt) of dry biomass in TSB controls to 39–66 % for PS, PE and PP thermally treated samples. The polymers obtained were analysed with nuclear magnetic resonance (NMR) and electrospray ionisation tandem mass spectrometry (ESI-MS/MS) to characterise their chemical structure. In conclusion, certain thermal treatment protocols of the waste plastics were shown to be viable for PHA production; with3-hydroxybutyrate and up to 12 mol% of 3-hydroxyvalerate and 3-hydroxyhexanoate co-monomeric units formed.
Direct proteolytic control of an extracytoplasmic function RNA polymerase sigma factor
Authors: Bilyk Bohdan , Kim Sora , Baker Tania , Seipke Ryan
Published: April 2019

Streptomyces species are well known for harbouring a large number of extracytoplasmic function (ECF) RNA polymerase sigma (σ) factors; nearly all of regulated genes required for morphological differentiation and/or response to environmental stress. The activity of ECF σ factors is typically modulated by a cognate anti-σ factor protein co-encoded at the same locus. In previous work, we identified σAntA as a cluster-situated regulator of starter unit biosynthesis in the production of antimycin, an anticancer compound. Unlike a canonical ECF σ factor, whose activity is controlled by a cognate anti-σ factor, σAntA is an orphan, which raises intriguing questions about how its activity is controlled. Inspection of the σAntA amino acid sequence revealed a carboxyterminal di-alanine, which is the only motif of the Clp-protease recognition signal obligately required for proteolysis. Here, we show by immunoblotting that the abundance of 3xFLAG-σAntA in vivo is enhanced by alteration of the C-terminal Ala-Ala to Asp-Asp and that abundance of both variants is elevated in the absence of genes encoding the Clp-protease and its regulatory subunits ClpX and ClpA. We also show that (His)6-SUMO-σAntA, but not a variant lacking the C-terminal di-alanine motif, is degraded by the ClpXP protease in vitro. These data unambiguously establish direct proteolysis as an alternative control strategy for ECF RNA polymerase σ factors and expands the paradigmatic understanding microbial signal transduction regulation.
Protein families are the punchline of the pangenomes
Authors: Crossman Lisa
Published: April 2019

Short-read draft whole genome assemblies can contain many contigs and be impacted on by repeat regions, such as those caused by mobile element activity or inherently repetitive gene structure. Annotating such assemblies for gene content and functional activity can be challenging. This can be especially true if the predicted genes are fragmented across contigs, very large, repetitive or of unusual nucleotide content. Very high and low %GC genomes also come with additional issues. The Pfam domain database 1 is a widely-used large collection of protein families, each represented by multiple sequence alignments and Hidden Markov Models (HMMs). Rather than studying the predicted whole gene content of draft genomes, or presence/absence of specific genes in pan-core genome analyses, we examined predicted protein content by Pfam domain complement. Here we present Punchline, a workflow written in Python 3, to study the genetic content of pangenome assemblies including draft assemblies by looking at the complement of short protein domains. The domains can be used in statistical comparisons of Bacterial groups of interest as provided to the workflow of Punchline. In addition, we show the application of Punchline to specific genomic data.

1. The Pfam protein families database in 2019: S. El-Gebali, J. Mistry, A. Bateman, S.R. Eddy, A. Luciani, S.C. Potter, M. Qureshi, L.J. Richardson, G.A. Salazar, A. Smart, E.L.L. Sonnhammer, L. Hirsh, L. Paladin, D. Piovesan, S.C.E. Tosatto, R.D. Finn Nucleic Acids Research (2019) doi: 10.1093/nar/gky995
Development of antimicrobials against Acanthamoeba
Authors: Carpinteyro Sanchez Alemao G. , Scott Fraser J. , Suckling Colin J. , Roberts Craig W.
Published: April 2019

Acanthamoeba is a free-living amoeba widely distributed in the environment which exists as two stages in their life cycle: a motile and trophic replicating trophozoite and a resistant cyst stage. In recent years, the incidence of infections due to Acanthamoeba spp. has shown a remarkable increase. This parasite is the causative agent of a sight-threatening infection of the cornea known as Acanthamoeba keratitis (AK) and a fatal disease of the central nervous system known as Granulomatous Amebic Encephalitis (GAE) mainly in immunocompromised patients. Although the trophozoite form is much more readily eliminated, at present there are not harmless effective treatments or a single drug that can eliminate both cystic and trophozoite forms. A particular set of compounds known as Minor Grove Binders (MGBs) have the characteristic of binding specifically to minor groove region of double-stranded DNA. The main effects of these MGBs are their ability to interfere with biological functions of DNA such as transcription machinery, also induction of apoptosis, hence cell death. These molecules have received great attention since they can be empirically screened and iteratively refined via chemical synthesis to target various entities such as tumors, bacteria, viruses and parasites. MGBs have been tested in vitrousing a colorimetric alamar Blue viability cell assayagainst Acanthamoeba castellanii Neff strain. To date, 2 hit compounds were able to inhibit trophozoites producing IC50s of 1.56 µM and 12.5 µM.
The Role of the cellular protein Dock5 as an egress restriction factor for Herpesvirus
Authors: Chadwick Ben , Schumann Sophie , Fuller Martin , Whitehouse Adrian , Fontana Juan
Published: April 2019

The herpesvirus family contains eight human pathogens that act as the causative agent of several human maladies. The herpesvirus family can be further subdivided alphaherpesviruses, betaherpesviruses, and the oncogenic gammaherpesviruses. Herpesvirus replication and capsid assembly occurs within the nucleus, from there capsids migrate into the cytoplasm. Following nuclear egress, viral capsids travel to the cellular membrane, where morphogenesis takes place. Recent analysis has demonstrated that the gammaherpesvirus Kaposi’s sarcoma associated herpesvirus (KSHV) upregulates the expression of a host miR-365 to target the cellular protein, DOCK5, to enhance KSHV egress. This hypothesis is supported by the observation that inhibition of miR-365 or overexpression of DOCK5 leads to the prevention of KSHV egress and accumulation of capsids at the plasma membrane. As herpesviruses share several mechanistic features, we are now employing electron microscopy to visualise the cytoplasmic capsid accumulation and to determine how DOCK5 is involved in herpesvirus egress. This will be achieved through advanced microscopy techniques such as correlative light microscopy, cryo-electron microscopy, and a novel nanobiopsy approach known as nanopipetting. Currently we have been establishing the optimal time of capsid egress in both KSHV and Herpes Simplex Virus-1 which will aid in visualising the process through transmission electron microscopy. A better understanding of herpesvirus egress will potentially help identify novel antiviral targets against herpesviruses.
Food inspection of the maltese cheeselet using hyperspectral imaging. The ‘food inspection using hyperspectral imaging’ (FIHI) project is financed by the Malta Council for Science and Technology, for and on behalf of the Foundation for Science and Technology, through the FUSION: R&I technology Development Programme
Authors: Griffin Sholeem , Falzon Owen , Camilleri Kenneth , Valdramidis Vasilis
Published: April 2019

The island of Malta has a rich heritage, which is evident in the rustic appeal of this island. Ġbejna forms an integral part of the Maltese food heritage. This artisan product is made from sheep or goat milk curds and aged for several months to develop its distinctive taste. During the ageing process, the cheese can become spoiled by fungi and unsafe for human consumption. This is a significant public health risk and a financial liability for producers. Conventional microbiology techniques do not detect these slow-growing pigment-less fungi early, allowing occasional distribution of contaminated products. We propose the use of hyperspectral imaging to detect these fungi during the early stages of cheese production. In contrast with a typical digital camera, which compiles the light signal into three broad wavelength bands; red, green and blue, a hyperspectral camera records numerous narrow and contiguous wavelength bands reflected from an object. This produces a series of images, each corresponding to the reflected electromagnetic energy in the respective narrow band of wavelengths. This image series may, in turn, be used for early fungal detection and identification. To test this hypothesis, a model cheeselet was produced to conduct compatibility and stability studies, through measurements of colony forming units, water activity, moisture levels, pH, protein and sugar content. The ġbejna model was then challenged with fungal strains isolated from commercial ġbejna and imaged using a hyperspectral camera. An algorithm is under development to differentiate contaminated samples from uncontaminated samples using image analysis and multivariate statistics.
Investigation on MFS family in C. parapsilosis with CRISPR
Authors: Zhao Fang , Butler Geraldine
Published: April 2019

C. parapsilosis causes candidiasis especially among newborn babies. The function of specific transporters is considered to be one key feature underlying drug resistance in Candida species. Drug transporters fall into two main classes – ATP-binding cassette (ABC) transporters, and the major facilitator superfamily (MFS). Particularly, Some members of members of the drug/H (+) antiporter family (DHA1) of the MFS superfamily function as multidrug transporters. We find that the DHA1 family in Candida species can be divided into several clades. These include MDR1/FLR1, associated with multidrug resistance in C. albicans (1 member in C. parapsilosis); TPO4, associated with polyamine transport (1 member in C. parapsilosis); NAG3/4, associated with transport of N-acetyl glucosamine (2 members in C. parapsilosis); TPO2/3, associated with polyamine transport (1 member in C. parapsilosis); YHR048w, with no known function (no members in C. parapsilosis); and TPO1/FLU1, possibly associated with fluconazole resistance (8 members in C. parapsilosis). We propose to use CRISPR-based gene editing to explore the function of all 13 members of the DHA1 family in C. parapsilosis. To date we have individually edited 10 members of the family by introducing stop codons near the start site of translation (ATG). We are currently editing the remaining 3 genes, and we are attempting to combine at least two edited genes in the same background. We will then test the phenotype of each edited strain in the presence of various drugs.
Comparative responses of paediatric airway epithelium to viral and allergen insult
Authors: Manzoor Sheerien , Broadbent Lindsay , McDermott Caoimhe , Main Stephen , Turner Steve , Schwarze Jurgen , Shields Michael , Power Ultan
Published: April 2019

Asthma affects over a million children in the UK. Early age viral infection, allergen sensitization, atopy and genetic predisposition increases the likelihood of developing asthma. Our aim is to understand the consequences of allergen exposure (house-dust mite; HDM) and/or RSV infection on innate immune responses, cytopathogenesis and virus replication in paediatric airway epithelium. Well-differentiated primary paediatric nasal epithelial cell (WD-PNEC) cultures derived from newborn or pre-school children were infected with RSV before or after stimulation with HDM. Virus growth kinetics, cytopathology and innate immune responses were analysed. HDM pre-treatment did not affect the culture integrity or RSV growth. Preliminary RT-qPCR analysis revealed upregulation of irf9 in RSV infected/HDM treated and HDM pre-treated/RSV infected cultures at 24 and 96 h post-infection (hpi). Isg15 was also upregulated in HDM pre-treated/RSV infected cultures at 24 hpi in newborns only. At 96 hpi, irf9, isg15, ifi6, duox2 and duoxA2 were upregulated in RSV infected cultures. Surprisingly, HDM treatment alone did not induce any significant upregulation of these genes compare to untreated/uninfected controls. However, data from more donors will be required to confirm these preliminary data. IL-29/IFNλ1 secretion was also significantly reduced following HDM pre-treatment of RSV infected cultures, while there was a trend towards reduced IL-6 secretion. These results suggest that HDM exposure modulates the innate immune responses to RSV infection of airway epithelium. The preliminary data are consistent with our over-arching hypothesis that our model of aeroallergen exposure and viral infection of airway epithelium will help elucidate mechanisms by which pre-school wheeze develops.
Lyme disease in Northern Ireland – a look back on serological testing
Authors: McCorry Louise , McCaughey Conall
Published: April 2019

Background

Lyme disease is an illness caused by the spirochete Borrrelia burgdoferi. NICE guidance suggests an approach to diagnosis resting on clinical presentation, presence in an epidemiologically plausible area, and positive serology. It is however, well documented that serological diagnostics in European Lyme disease is problematic. There is a movement for Lyme advocacy, a voice that emphasises the poor diagnostics in this infection. With the spotlight on Lyme disease, we are increasingly seeing patients presenting in Northern Ireland across a broad range of specialities. Currently there is minimal data looking at the local epidemiology of Lyme disease. We present previously unpublished data looking at the demographics of those testing positive for Borrelia burgdoferi antibodies.

Methods

We collected data from all patients tested for Lyme disease in Northern Ireland from 2013–2018. We excluded negative results. A randomised selection of 300 cases were chosen. We recorded patient age, gender, origins of testing, home postcode, tick bite history, relevant travel history, duration of symptoms, timings of testing, antibody results including confirmatory testing and treatment.

Results

Of the 300 selected patients 241 (80 %) were IgM positive and 83 (30 %) IgG positive using DiaSorin, Liason CLIA. The average age of presentation was 39 years old. There was a predominance of females tested (62%). 32 % of tests originated in primary care. Of the IgM positives only 41 (17 %) were confirmed by C6 peptide or lineblot in the reference laboratory (RIPL, Porton Down). The results highlight the likely low seroprevalence of Lyme disease in Northern Ireland.
A possible alternative for chromosome dimer resolution in E. coli
Authors: Croft Cameron , Macpherson Cindy , Summers David
Published: April 2019

Dimer formation is a serious threat to the stable maintenance of ColE1-like plasmids. Dimers form infrequently by homologous recombination but accumulate rapidly by having two origins of replication. This results in elevated plasmid loss and a reduction in host cell growth rate. Plasmid dimers are resolved to monomers by the XerCD recombinase plus accessory proteins ArgR and PepA, acting at the cer recombination site. The circular chromosome of E. coli also forms dimers infrequently, and consequent failure of chromosome partition results in filamentation, SOS induction, and failure of cell division. Site-specific recombination is required for dimer resolution during cell division in a process facilitated by XerCD acting at the dif (deletion induced filamentation) site near the E. coli chromosome terminus. ArgR and PepA accessory proteins are nonessential, but the septum-associated protein FtsK is necessary for dimer segregation, suggesting the XerCD/difcomplex interacts with division septums. Our preliminary work had revealed homology between cer and a 170 bp chromosomal site (tcs, terminal region cer-like site) 1.2 min from dif. The tcs site and surrounding region was cloned into plasmids, dimer plasmids were purified and then assayed for tcs-mediated recombination. Our results demonstrate that a construct with a 500 bp tcs insert supports XerCD-mediated recombination, whereas smaller constructs were recombination-deficient. The absence of plasmid monomerization in mutant strains indicates that ArgR and PepA are required for recombination. Additionally, the tcs knockout strain displayed moderate filamentation of cells. Given the similarities between cer, tcs, and dif, these results suggest that tcs could facilitate chromosome dimer resolution.
Investigating the role of low-oxygen-activated (lxa) encoded proteins in the pathogenesis of Burkholderia cepacia complex and their contribution to chronic infection in cystic fibrosis
Authors: O’Connor Andrew , McClean Siobhán
Published: April 2019

Burkholderia cepacia complex (Bcc) is a group of 22 closely related species of Gram-negative bacteria that cause chronic infections in people with cystic fibrosis (CF) that are rarely eradicated. The high-level antibiotic resistance and poorly understood mechanisms by which Bcc survive and persist during chronic infection mean that combatting these chronic infections is particularly challenging. We have previously found that a group of 20 low-oxygen-activated (lxa) encoded proteins were consistently upregulated in sequential Bcc isolates in two chronically infected CF patients over time of infection. Many of these proteins have not previously been studied in Bcc but the consistent upregulation over time of infection suggests that they play an important role during chronic infection. Two particular proteins of interest within the lxalocus that were consistently upregulated were a universal stress protein (USP) and a phospholipid binding protein (PBP). Single gene deletion mutants (Δusp and Δpbp) in the wildtype Bcc strain K56-2 (WT) both showed a 90 % reduction in attachment to CFBE41o- cells compared to WT. There was also a 5-fold reduction in the virulence of Δpbp in the acute infection model Galleria mellonella (P<0.005) and an increased sensitivity of Δusp to peroxide-induced oxidative stress (P<0.0001) and low pH (P<0.05) relative to WT. A reduction in both uptake and survival of Δusp in U937 macrophage-like cell line compared to WT, suggests the USP plays a role in the intra-macrophage survival of Bcc. Overall, these proteins, previously associated with low-oxygen conditions may play a considerable role in Bcc pathogenesis and its adaption during chronic infection.
Low prevalence of resistance mutations to integrase inhibitors (INI) in patients living with HIV-1 at baseline using next generation sequencing (NGS)
Authors: Hubb Jon , Hopkins Mark , Deayton Jane , May Sophie
Published: April 2019

Current guidelines indicate that baseline INI resistance testing is not required; however, increased prescribing may lead to increased resistance mutation prevalence. NGS allows the detection of minority variants (MVs) not be detected by conventional methods. Barts Health NHS Trust patient records were reviewed to identify INI naïve patients prescribed INI between March 2014 and March 2017. Baseline plasma samples were extracted for nucleic acid and amplified in a single RT-PCR. Libraries were prepared using the Nextera XT library preparation kit and sequencing performed on an Illumina Miseq. Sequencing reads were assembled into consensus sequences and resistances determined using the Stanford HIV resistance database (HIVdb). MVs were variants <20 % of the overall population. Of 248 patients prescribed INI, 128 patients were excluded from the study due to an undetectable viral load or lack of sample. Of the remaining 121 patients, integrase sequences were successfully obtained from 91. No major INI mutations were detected. Six patients were identified with an HIVdb INI score ≥10: E157Q (4 patients) and T97A (2 patients). Only 1 MV accessory mutations was observed (A128T). Of patients with resistance mutations, 5 received raltegravir- and 2 received dolutegravir-based regimes. No treatment failures were observed. Prevalence of baseline INI mutations increased in recent years: 0/12 (0 %) patients in 2014–2015, 2/32 (6.3 %) patients in 2015–2016 and 5/47 (10.6 %) patients in 2016–2017. Resistance MVs to INI were extremely rare. Although this study supports current baseline INI resistance testing guidelines, increasing mutation prevalence’s may require future revision of guidelines.
Investigating soil bacteria for novel antimicrobials which inhibit the opportunistic pathogen, Staphylococcus epidermidis
Authors: Twomey Ellen , Fields Des , Begley Maire
Published: April 2019

According to the European Center of Disease Control and Prevention, Staphylococcus epidermidis is rapidly becoming a serious concern in hospitals as a cause of resistant infections, particularly in the case of in dwelling and prosthetic medical devices. To overcome this issue, new, potent antimicrobials with novel target pathways need to be uncovered to both reduce morbidity and mortality, and the spread of resistant microbes. 51 bacterial strains with observable antimicrobial activity against S. epidermidis were isolated from samples of soil, collected from various locations around Ireland. The activity was reconfirmed, and the most effective strains were shortlisted using deferred antagonism assays. Characterisation tests, (Gram stains, oxidase and catalase testing and 16S sequencing), were carried out to determine the identities of the bacteria at hand. Presently studies are underway to elucidate the nature of the antimicrobial activity and to govern if the findings are novel. HPLC is being used to purify the compounds, with proteinase K assays evaluating if the antimicrobials are proteinaceous in nature. The future of this study will include the sequencing of any uncovered antimicrobial peptides, MIC determination of antimicrobial compounds, observing the impact on biofilm formation and looking at potential efficacy against other relevant pathogens. With the identification of a novel compounds, this study aims to present an opening into potential new treatment options to help address the current struggle against antimicrobial resistance.
Oxford Nanopore sequencing elucidates a novel stx2f carrying prophage in a Shiga toxin producing Escherichia coli(STEC) O63:H6 associated with a case of haemolytic uremic syndrome (HUS)
Authors: Greig David , Dallman Timothy , Jenkins Claire
Published: April 2019

Aim

The aim of this study was to use Oxford Nanopore sequencing to characterise a Shiga-toxin producing Escherichia coli(STEC) O63:H6 responsible for a recent case of haemolytic uremic syndrome (HUS).

Methods

STEC isolate 377323 and a comparator stxnegative E. coli isolate of the same lineage and serotype (382634) were sequenced on an Oxford Nanopore minION R9.4 flow cell and an Illumina HiSeq 2500. Nanopore FAST5 data were basecalled and demultiplexed by Albacore. Assemblies were generated using Unicycler and polished using Nanopolish, Pilon and Racon before screening for prophages using PHASTER.

Results

The Shiga toxin subtype was confirmed to be stx2f, which is rarely associated with the onset of HUS in STEC. Comparing the two genomes, the stxnegative isolate harboured five prophages compared to eleven prophages in the STEC isolate, with only one in common. Both samples contained an intact locus of enterocyte effacement pathogenicity island. The Shiga toxin encoding prophage was 42.5 kb in size and predicted to contain 71 coding sequences. 377323 also encoded an 85kbp IncFIB plasmid, not present in 382634. This plasmid encodes a traconjugation cassette and the virulence bfpcassette encoding for a type IV bundle forming pili.

Conclusion

Long read sequencing enabled the characterisation of a rare STEC and related E. coli IN relation to both prophage content and plasmid content and revealed significant differences in the pan-genome in addition to the acquisition of the novel Stx2f phage. This data contributes to the understanding of non-O157 STEC associated with HUS.
Validation of a hepatitis B virus (HBV) Next Generation Sequencing (NGS) pipeline at Barts Health NHS Trust
Authors: Hubb Jon , Hopkins Mark , May Sophie , Jeffery-Smith Anna
Published: April 2019

At Barts Health NHS Trust, historically HBV genotyping and resistance testing is performed using nested PCR followed by Sanger sequencing. With the development of NGS assays for HIV and HCV, in order to harmonise workflows an NGS HBV pipeline was developed. Primers were designed for the whole genome amplification of HBV. Plasma samples were extracted for nucleic acid using a Qiagen Qiasymphony. Whole genomes of HBV were amplified in a single round PCR and quantified using a Qubit fluorometer. Libraries were prepared using the Nextera XT library preparation kit and sequencing was performed on an Illumina Miseq. Sequencing reads were assembled into consensus sequences using a Linex-based pipeline. Resistances and genotypes were determined using the HBV Grade website. Minority variants were variants <20 % of the overall population. Pan-genotypic PCR primers were designed in the nick region of the partially double stranded HBV genome. In-silico analysis allowed primers to be designed to bind multiple genotypes. Sensitivity analysis of primers was investigated for the most common circulating genotypes. A panel of HBV positive and external quality assurance samples showed good comparability to Sanger sequencing results. Inter- and intra-assay variability showed the assay was robust and fit for purpose. No resistance associated minority variants were observed. The development an NGS pipeline for analysis of HBV allowed the harmonisation of diagnostic pathways within the virology sequencing laboratory. Introduction of this test would allow data to be gathered to assess the importance of minority variants in HBV infection.
Mycovirus induced hypervirulence of Leptosphaeria biglobosa enhances systemic acquired resistance to Leptosphaeria maculans in Brassica napus
Authors: Shah Unnati , Kotta-Loizou Ioly , Fitt Bruce , Coutts Robert
Published: April 2019

Phoma stem canker is one of the most important diseases of winter oil seed rape (Brassica napus) world-wide and is caused by a complex that comprises at least two species: Leptosphaeria maculans and Leptosphaeria biglobosa. Screening a panel of field Leptosphaeria isolates from B. napus for the presence of mycoviruses revealed the presence of a novel double stranded (ds) RNA virus in L. biglobosa and no viruses in L. maculans. The virus forms isometric particles ca. 40–45 nm in diameter and has four genomic segments, each possessing a single open reading frame flanked by untranslated regions. Phylogenetic analysis revealed modest similarities to known and suspected members of the family Quadriviridaeand therefore the virus was nominated Leptosphaeria biglobosa quadrivirus-1. Following eradication of the mycovirus, virus-infected and virus-free isogenic lines of L. biglobosa were created. A direct comparison of the growth and virulence of these isogenic lines illustrated that virus infection caused hypervirulence and resulted in induced systemic resistance towards L. maculans in B. napus following lower leaf pre-inoculation with the virus-infected isolate. Analysis of the plant transcriptome suggests that the presence of the virus leads to subtle alterations in metabolism and plant defences. For instance, transcripts involved in carbohydrate and amino acid metabolism are enriched in plants treated with the virus-infected isolate, while pathogenesis-related proteins, chitinases and WRKY transcription factors are differentially expressed. These results illustrate the potential for deliberate inoculation of plants with hypervirulent L. biglobosa to decrease the severity of phoma stem canker later in the growing season.
Inhibition of IFN-gamma signalling by vaccinia virus protein C6: a multi-functional interferon antagonist
Authors: Talbot-Cooper Callum , Lu Yongxu , Stewart Jennifer , Smith Geoffrey
Published: April 2019

Vaccinia virus (VACV) encodes multiple proteins that function to inhibit both the production and downstream effects of interferons, which are critical modulators of the antiviral response. One such protein, VACV C6, inhibits both the production of type I IFN (by blocking IRF3 signalling) and the downstream effects of type I IFN (by blocking JAK-STAT signalling). Further to this, our findings suggest C6 can also inhibit the downstream effects of type II IFN of which IFN-gamma is the sole member. The observation that C6 co-immunoprecipitates with STAT1, a protein crucial in activating the downstream functions of IFN-gamma, might explain its inhibitory activity.
Use of Bacteroides-derived microvesicles for mucosal vaccines
Authors: Miquel-Clopes Ariadna , Carvalho Ana L. , Wegmann Udo , Bentley Eleanor G. , Al-Katy Sanaria H.M. , Coombes Janine L. , Kipar Anja , Stentz Regis , Stewart James P. , Carding Simon R.
Published: April 2019

The vast majority of infectious agents enter the body via mucosal sites yet there are very few licensed mucosal vaccines able to generate protective immunity at the sites of pathogen entry. A major obstacle in developing mucosal vaccine is delivering biologically active vaccine antigens (Ag) to mucosa-associated lymphoid tissues to prime protective immune responses. To address these issues we have developed a drug delivery technology platform to deliver intact antigens to the respiratory and gastrointestinal tract using outer membrane vesicles (OMV) naturally produced by Bacteroides thetaiotaomicron (Bt), a non-pathogenic human commensal gut bacterium. We have developed the capability of engineering Bt to express antigens of interest in their OMVs which we have shown are stable for long periods of time across a wide temperatures range. They also have inherent adjuvanticity as shown by the ability of native OMVs to elicit the formation of organised lymphoid follicles comprising dendritic cells and T and B cells in both the upper and lower respiratory tract after intranasal administration. The intransal administration of Bt OMVs expressing the pre-fusion headless hemagglutinin mini-stem protein of influenza type A virus (IAV) subtype, H5N1, induced high titre antigen-specific antibodies in the respiratory mucosa (IgA) and serum (IgG) that conferred heterotypic protection to infection by a H1N1 IAV. Collectively, our data demonstrates the feasibility of using Bt OMVs in mucosal vaccine formulations for respiratory infections.
Metabolic profiling and environmental characterisation of salterns in the islands of Cabo Verde
Authors: McCulloch Bryn , Rowson James , da Mour Aires , Rocha Hélio , Filipa Simões Marta , Antunes André
Published: April 2019

The Cabo Verde Islands constitute a biodiversity hotspot, encompassing a wide range of different biotopes. Thus far, most surveys and programmes have focused exclusively on the flora and/or fauna of the islands and no effort has been made to systematically assess and preserve locally existing microbial biodiversity. The absence of such studies is especially troubling regarding their traditional salterns, as many of them have been abandoned, and increase of construction in coastal areas has already started to indelibly change some of these unique ecosystems and will likely obliterate some of them. Here we provide an overview of the preliminary data recently collected from salterns in the islands of Sal, Maio, and Boavista and include details on their previously unreported physical-chemical characteristics (salinity, pH, temperature and ionic composition), as well as first results on microbial metabolic profiling. Our data has shown a wide diversity of environmental niches (particularly noticeable in Maio), and showcased differences in substrate use both between and within different salterns. These are the first step of our ongoing efforts in assisting in the survey of several threatened extreme environments in Cabo Verde, characterising their geochemistry and microbiology, and identifying their potential biotechnological applications.
Zika virus: persisting tissue reservoirs and their potential for clinically relevant pathology
Authors: Ferguson Debbie , Hall Jo , Ham Claire , Jenkins Adrian , Giles Elaine , Kempster Sarah , Berry Neil , Almond Neil
Published: April 2019

The rapid spread of Zika virus through the Caribbean and Americas appears associated with greater levels of congenital zika and Guillain-Barre syndromes than previously seen. In addition, new disease pathologies such as persisting sexual transmission and severe liver injury have been identified. With zika vaccines years away from licensing an understanding of potential long-term health consequences following adult infection needs to be gained, especially where organs from infected people may be used for transplantation. To model the pathology of human Zika virus infection Old World and New World non-human primates were sub-cutaneously infected with a Caribbean Zika virus isolate. Following termination during primary viremia or later time points post peripheral viral clearance FFPE tissue sections were analysed for Zika virus RNA (RNAscope) and host responses (immunohistochemistry). Zika virus RNA was detected in multiple tissues from 3dpc with low levels of viral RNA detectable by RNAscope through to 101 dpc. New World non-human primates retained higher levels of persisting virus within tissues, notably within spleen, small intestine, kidney, liver and genital tissues. Differing pathologies were observed with Zika virus detected clustered within kidney glomeruli and associated with liver inflammatory infiltrates within New World species. Such pathologies are being documented in adults with resolved primary infection symptoms. This includes clinical complications following solid organ transplantation where immune suppression may allow viral reactivation either from the patient or transplanted organ. In the absence of a protective vaccine the potential risks from persisting viral reservoirs needs to be understood to ensure appropriate clinical management.
Characterising the genomes and transcriptomes of hyper ammonia producing bacteria from the rumen
Authors: Friedersdorff Jess , Creevey Chris , Kingston-Smith Alison
Published: April 2019

Ruminants depend on the highly diverse microbial community that resides in the rumen, the first and largest chamber of their digestive system, to gain nutrients from their herbivorous diet. The Hyper Ammonia Producers (HAPs) are obligate amino acid fermenting bacteria found in low numbers in this community. This break down of amino acids and peptides results in excessive ammonia production, as well as hydrogen and carbon dioxide, resulting in loss of nitrogen from the host and contribution to environmental emissions from enteric fermentation. Despite their large impact, little is known about the genomic underpinnings of the HAP phenotype. Our study employed comparative genomics and transcriptomics approaches to address this question. A phylogenetic tree of 498 rumen prokaryotic microbial genomes from the Hungate 1000 project (including 12 known HAPs) identified the HAP phenotype as polyphyletic, indicating independent origins or a result of horizontal gene transfer (HGT). However, following construction of sequence similarity networks for all genomes, few uniquely shared homologous genes families were apparent in the HAPs, suggesting that HGT did not drive their evolution. Instead, independent evolution of the phenotype is supported by similar functional analog profiles in the genomes of organisms with the HAP phenotype. Genome-wide characterisation and expression of functional analogs in known HAPS will allow in silico prediction of novel HAPs from the rumen which can be confirmed in vitro.
Investigating the requirement for host cell chloride ion channels during human respiratory syncytial virus infection
Authors: Pearson Hayley , Hover Samantha , Todd Eleanor , Lipiatt Jonathan , Barr John , Mankouri Jamel
Published: April 2019

Ion channels are a diverse class of transmembrane proteins, which selectively allow ions across cellular membranes, influencing a multitude of cellular processes. Modulation of these channels by viruses is emerging as an important host-pathogen interaction, and has been demonstrated to regulate critical stages of the virus multiplication cycle including entry, replication and egress. Human respiratory syncytial virus (HRSV) causes severe respiratory tract infections (RTIs) globally and is one of the most lethal respiratory pathogens for infants in developing countries, with many cases leading to severe lower respiratory tract infections, and the development of bronchiolitis. Evidence also suggests that childhood HRSV infection contributes towards the increased incidence of adult asthma. There is no HRSV vaccine, and the only treatment is immunoprophylaxis that is prohibitively expensive and only moderately effective; thus new treatment options are required. In this study, by infecting human lung epithelial cells with HRSV in the presence of various broad-range channel modulators, Cl- channels were identified to play an important role during HRSV infection. Time of addition assays using these broad-acting Cl- channel blockers identified the stages within the HRSV lifecycle that were dependant on Cl- channel activity, and the use of family-specific Cl- channel blocking drugs identified a small sub-family of Cl-channels which, when inhibited, resulted in significantly reduced HRSV multiplication. We are now identifying the specific Cl- channel(s) facilitating the multiplication of HRSV using genetic means, and well as assessing the importance of Cl- channels in replication cycles of other negative sense RNA viruses.