- Volume 1, Issue 1A, 2019
Volume 1, Issue 1A, 2019
- Poster Presentation
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- Virology Workshop: Morphogenesis, Egress and Entry
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An exploration of antigen expression of hepatitis C entry receptors on equine cells in relation to equine hepacivirus A
More LessEquine hepacivirus A (EqHV) belongs to the family Flaviviridae and has been identified as the hepacivirus phylogenetically most closely related to Hepatitis C virus (HCV). Like HCV, EqHV is a hepatotropic virus that has been reported in over fourteen countries from six continents. It has a high prevalence in some populations, in particular the Thoroughbred breed. However, much is still unknown about EqHV, such as its pathogenesis and mode of transmission. The main four receptors used by HCV for viral entry to human hepatocytes are Cluster of Differentiation-81 (CD-81), occludin (OCLN), claudin-1 (CLDN-1) and Scavenger Receptor Class B Member 1 (SCAR-B1). This study investigated the distribution and expression of these entry receptors on equine cells by flow cytometry, immunocytochemistry and immunohistochemistry. Using a human liver cell line (Huh-7) as a positive control, antibodies against HCV receptors appeared to cross-react with antigens on equine cells. The proportion of fetal horse kidney cells that expressed CD-81, OCLN, and CLDN-1 was 37.2 %, 16.0 %, and 7.0 %, respectively, whereas the equine dermal cells (E.Derms) expressed CD-81 (96.0 %), OCLN (1.2 %) and CLDN-1 (1.7 %). CD-81, OCLN and CLDN-1 were also expressed on E.Derms and Huh7 cells, as detected by immunocytochemistry and on equine liver cells and the allantochorionic region of Thoroughbred placenta by immunohistochemistry. These findings form the basis for further comparative investigation into the entry receptors used by EqHV to infect equine and human cells. Such information may inform future studies on EqHV pathogenesis and mode(s) of transmission.
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High throughput screening to identify host-factors involved in RSV fusion
More LessRespiratory syncytial virus (RSV) is the major cause of childhood respiratory disease; however, currently there is no licenced vaccine available and the only therapeutic, a monoclonal antibody against the viral Fusion (F) protein, is expensive and applied sparingly. RSV particles enter cells by membrane fusion, orchestrated by F – a type I integral membrane protein. This process was recently shown to involve macropinocytosis of the particle. Separately, RSV can spread through induction of direct cell-cell fusion – again orchestrated by F. Little is currently known about the host-factors involved in regulating or inhibiting RSV F-mediated fusion. Here, using two different high-throughput screening approaches, we have identified host-factors involved in regulating RSV fusion. Using quantitative mass-spectrometry analysis of isolated cell membrane fractions from mock and RSV-infected cells we have identified membrane proteins which are differentially regulated during RSV infection. Furthermore, using lentiviral libraries expressing individual interferon stimulated genes (ISGs) from different mammalian species we have investigated ISG-mediated inhibition of RSV fusion. Our data provides important insights into host-factors involved in RSV spread, furthering our understanding of the fusion process and identifying potential targets for antiviral therapy.
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The Role of the cellular protein Dock5 as an egress restriction factor for Herpesvirus
More LessThe 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.
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Production of Influenza C HEF pseudotyped lentiviral particles
More LessWhile influenza A and B virus contain the two glycoproteins Hemagglutinin (HA) and Neuraminidase (NA) inserted into the viral membrane, influenza C virus possesses only one spike designated Hemagglutinin-Esterase-Fusion (HEF) protein which combines the functions of both HA and NA. Like HA, it recognizes and binds to a receptor on the cell surface to initiate virus entry. However, the receptor is N-acetyl-9-O-acetylneuraminic acid. HEF is the receptor-destroying enzyme, which is the function of the neuraminidase (NA) in influenza A and B virus. Although Influenza C virus infections are generally mild and self-limited, it comprises around 13 % of influenza positive cases in hospitalized children. It is also a cause of community acquired pneumonia of children. As influenza research has been more focused on Influenza A and B, it is of interest to pseudotype influenza C HEF. The full sequence of Inflenza C HEF (C/Tokyo/4/2014) was obtained and cloned into pi.18 expression plasmid. The second generation packaging construct pCMVΔR8.91, and the self-inactivating lentiviral vector pCSFLW expressing firefly luciferase, and pCSGW expressing GFP were used for lentiviral vector particle production. The original HEF sequence was mutated by site directed mutagenesis to inhibit esterase activity to prevent receptor destruction prior to pseudotype viral entry. Pseudotyped particles bearing the HEF glycoprotein were successfully produced and verified by fluorescence and Luciferase titration assay with titres of 5×107 RLU/ml. Currently, optimization is undergoing to achieve higher titres as well determining the specific protease/s utilized by HEF for its activation.
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- Virology Workshop: Pathogenesis
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Identifying the role of complement receptor 2 (CR2) on follicular dendritic cells (FDCs) in the persistence of foot and mouth disease virus (FMDV)
More LessMore than 50 % of cattle (regardless of foot and mouth disease (FMD) vaccination) become persistently infected for long periods of time (years) after exposure to FMD virus (FMDV). The mechanisms associated with establishment of persistent infections are still poorly understood. I am testing the hypothesis that complement receptor 2 (CR2) on follicular dendritic cells (FDCs) located in the germinal centers of the lymphoid tissue, are involved in the trapping and long-term persistence of FMDV and that this persistence is essential for the maintenance of long-term antibody responses to FMDV. The aim of this study was to assess FMDV antigen retention and the generation of the specific immune response in vivo, in a mouse model. Groups of mice were treated with an anti-CR2 monoclonal antibody, named 4B2, which has been described previously to block CR2 long-term in vivo, blocking for 6 weeks after a single injection of 2 mg (Kulik et al., 2015). After treatment with 4B2, animals were infected with FMDV and lymphoid tissues and serum samples evaluated for the presence of antigen and the humoral immune response, respectively. The ability of 4B2 to block the binding of FMDV and PAP immune complexes (ICs) to FDCs in vitro as well as results about the role of FDC in trapping FMDV via CR2 in vivo is currently being investigated and will be discussed.
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Studying the mammalian adaptation potential of influenza A viruses in a single infection cycle
More LessVarious influenza A viruses (AIVs) have infected humans in the last decade. These infections result from spill over into humans at times of AIV poultry outbreaks with severe clinical consequences for those infected. These AIVs do not have the ability to be efficiently spread among humans via respiratory droplets or aerosols and subsequently cause a pandemic. However, adaptation of these avian influenza viruses to humans by mutation or reassortment may change this. We know that AIVs require adaptation of several critical characteristics in order to effectively spread between humans. Therefore, in order for human pandemic emergence, AIV that crosses into a human host must already be sufficiently able, or must rapidly adapt to overcome these constraints to transmit to a subsequent human host before infection is cleared by an immune response. In this study, we used mice and analysed the mutations acquired by avian origin H7N9 and H9N2 isolates in a temporal fashion during a single infection cycle in individual hosts. Viruses recovered from infected mice lungs were subjected to Next Generation sequencing (NGS) to identify mammalian adaptation enhancing mutations. Moreover, the timing of these mutations was correlated to the host response, with particular emphasis on cytokine profile and innate immune responses during the time prior to and following viral sequence changes to help understand the virus-host dynamics and the host environment favouring adaptation. Furthermore, understanding of the determinants and mechanisms of adaptation and transmission may aid in assessing the risks posed by avian influenza A viruses to human health.
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Detection of influenza D virus in respiratory disease samples from Northern Irish cattle
Influenza D virus (IDV) is a newly described member of the Orthomyxoviridae family, initially identified during a 2011 outbreak of respiratory disease in North American pigs. Cattle were subsequently shown to be the main reservoir of the virus and accumulating evidence suggests a role for IDV in bovine respiratory disease complex. During the winter of 2017/2018, cattle submitted to the Agri-Food and Biosciences Institute for post-mortem with confirmed respiratory disease were tested for the presence of IDV by real-time RT-PCR. Virus isolation was performed in Swine Testes cells and full-genome sequence determined. Of 104 cattle with confirmed respiratory disease, 9 tested positive for IDV (8.7 % prevalence). Virus was detected in both the upper and lower respiratory tract. Lung tissues from IDV positive samples were negative for the presence of bovine herpesvirus 1, bovine respiratory syncytial virus, bovine viral diarrhea virus and parainfluenza virus 3. Of the 9 cattle which tested positive for IDV, 3 tested positive for coronavirus. Histological analysis of lungs from IDV positive samples revealed pathological features including necrosis, neutrophil infiltration of alveolar spaces, fibrosis, congestion, oedema and haemorrhage. Sequenced isolates were shown to cluster with European isolates of the D/swine/Oklahoma/1334/2011 clade. To date, IDV has been detected in North America, Mexico, Japan, China, France, Italy and the Republic of Ireland. This study is the first to identify IDV in UK cattle herds. The presence of IDV in respiratory disease samples supports a role for this virus in bovine respiratory disease complex.
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BTV-GLUE: a new bioinformatic resource for genomic studies of Bluetongue virus
Bluetongue virus (BTV) is an arbovirus transmitted by biting midges (Culicoides pp.). BTV causes a severe disease (bluetongue) in domestic and wild ruminant species with high levels of morbidity and mortality. Bluetongue has emerged as an important disease in sheep and cattle worldwide. The BTV genome is composed by ten linear dsRNA segments, packaged within a triple-layered icosahedral protein-capsid, and encode 7 structural and 4/5 non-structural proteins. To date, there are at least 27 BTV serotypes (mainly determined by the VP2 outer capsid protein) circulating worldwide. In addition, high rates of reassortment involving all genome segments have been documented, complicating epidemiological studies and vaccination programmes. We have developed BTV-GLUE (http://btv.glue.cvr.ac.uk), a new bioinformatics sequence data resource for bluetongue virus. Sequences from the NCBI nucleotide database are curated along with complementary sequence metadata and are integrated together inside GLUE (http://tools.glue.cvr.ac.uk), a data-centric software package for capturing virus sequence data and organising it along evolutionary lines. The dataset also contains reference sequences with genome feature annotations, multiple sequence alignments, defined clades and phylogenetic trees, for each BTV segment and clade. A new automated genotyping tool for all segments has been developed. The resource may also be used as an offline bioinformatics toolkit. BTV-GLUE will help the BTV community to study varying aspects of BTV biology and evolution and will facilitate the adoption of a nomenclature that more easily distinguishes the properties of BTV strains circulating worldwide.
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Developing a pseudotyping assay for Zika virus glycoprotein
More LessZika 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.
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Exploring the molecular inter-relationship of SRPK1 and SRSFs during Human Rhinovirus (HRV)-infection and their subsequent effects on viral replication
More LessIntroductionHRV infections cause asthma exacerbations, while HRV-induced wheezing in pre-school children is associated with asthma development in later life. Molecular mechanisms underlying this pathophysiology remain elusive, while HRV-C is increasingly associated with disease severity. Transcriptomic analysis of human respiratory (nasal) epithelia from an in vivo HRV-C infection model revealed changes in expression of cellular Serine/Arginine Protein Kinase 1 (SRPK1) and its substrates, the Serine/Arginine Splicing Factors (SRSFs) suggesting that HRV infection targets pre-mRNA splicing and/or its regulating factors.
Hypothesis1) HRV splicing modulation results in host transcriptome changes, leading to impaired anti-viral responses and/or immunoregulation; 2) HRV-induced changes to SR proteins result in enhanced HRV-translation and replication.
MethodsHRV-A RNA was synthesised by in-vitro transcription from pRV16.11. HRV replication was measured by RT-qPCR and titration in HeLa cells. Protein expression was monitored by western blotting and confocal microscopy. SPRIN340 was used as a specific SRPK1 inhibitor. Alternatively, SRPK1 was depleted via siRNA interference.
Results1) SRPK1, SRSF1-6 and SRSF9 are expressed in both A549 and HeLa cells; levels were greater in the latter. 2) SRPIN340 and siSRPK1 treatments decrease the levels of SRPK1 leading to decreased expression of SRSFs in HeLa and A549 cells. 3) HRV16 infection decreased SRPK1, SRSF1-6 and SRSF9 expression in HeLa and A549 cells.
ConclusionOur initial data suggest that HRV-infection specifically alters molecules involved in pre-mRNA splicing, providing us with insights into the molecular mechanisms underlying the distinct pathology of HRV infection, as well as the aetiology of HRV-associated asthma.
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Aberrant RNA replication products of highly pathogenic avian influenza viruses and its impact in the mammalian associated cytokine storm
More LessHighly 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.
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Human immunodeficiency virus type 2 (HIV-2) dynamics and whole genome deep sequence analysis in Mauritian-origin cynomolgus macaques (Macaca fascicularis)
More LessHuman 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.
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Infectious bursal disease virus (IBDV) replicates in the gut associated lymphoid tissue and alters the gut microbiome of chickens
More LessInfectious bursal disease virus (IBDV) is a member of the Birnaviridae family that infects B cells in chickens, leading to immunosuppression and mortality. Immunosuppression is known to exacerbate the colonisation and shedding of zoonotic gut bacteria, for example Campylobacter jejuni, Salmonella Enteritidis and Escherichia coli, for reasons that are poorly understood. In order to address this, we infected groups of chickens (n=6) with either a classical strain (F52-70) or a very virulent strain (UK661) of IBDV. At 3 days post-infection, both strains were found to replicate in the gut-associated lymphoid tissue of thecaecaltonsils. 16 s rRNA sequencing revealed that in birds infected with IBDV, regardless of strain, there was a decrease in bacterial diversity in the caecal tonsils, but an increase in diversity of bacteria shed from the cloaca. Secretary IgA binding to commensal bacteria is known to influence the composition of the microbiome, and we speculate that IBDV alters the repertoire of sIgA thereby altering the microbiome composition. Interestingly, we found the number of clostridial species was reduced following IBDV infection. Clostridial species have been shown to induce Treg populations in mice and we speculate that IBDV-mediated changes in the microbiome affect the population of different immune cells in the mucosa. Taken together, we hypothesise that IBDV infection directly affects the B cell population and indirectly affects other immune cell populations in the gut and alters the gut microbiome, which leads to a more favourable environment for zoonotic bacterial infections to colonise.
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Zika virus: out of sight but not out of the mind. Viral persistence and host responses within the central and peripheral nervous systems
Following the rapid spread of Zika virus infection through the Caribbean and Americas, model systems of Zika virus infection have been established to determine disease pathology and potential health impacts for those infected with this neurotropic flavivirus. NIBSC has established both Old World (rhesus and cynomolgus macaques) and New World (red-bellied tamarins) non-human primate disease models of human Zika virus infection. Animals were infected sub-cutaneously with the Caribbean Zika isolate PRVABC59 and pairs terminated during primary viremia (3 dpc) or post clearance of plasma viremia (42 and 101 dpc). FFPE sections from multiple tissues, including brain and peripheral nerves were analysed for Zika virus RNA (RNAscope), viral proteins and host responses (immunohistochemistry). All animals became infected and rapidly cleared high levels of peripheral viremia. Zika virus RNA was detected in multiple tissues from 3 dpc with low levels of viral RNA and viral NS1 protein remaining detectable through to 101 dpc. Viral RNA was associated with both glial and neuronal cells throughout the brain and schwann cell nuclei in peripheral nerves. Neuroinflammation (astrogliosis, microgliosis, peri-vascular cuffing) and increased levels of CD3+, CD8+ or CD45+ cells were present from 3 dpc through to 101 dpc. Disruption of MAP2 neuronal dendrite staining and peripheral nerve myelin basic protein staining was also observed. The continued detection of Zika virus within tissues 3 months post infection and in the absence of a detectable peripheral viremia raises questions regarding potential long term effects of this virus and associated inflammatory responses within the nervous systems.
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Zika virus: persisting tissue reservoirs and their potential for clinically relevant pathology
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.
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Investigating the requirement for host cell chloride ion channels during human respiratory syncytial virus infection
More LessIon 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.
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Unravelling the features of Influenza as entry mechanism
Influenza virus is the causative agent of the ‘flu’. According to the World Health Organisation, Influenza causes up to 5 million cases of severe flu and 500 000 deaths annually. To release its genome inside the cell and start an infection, Influenza virus must fuse its envelope with the endosomal membrane of the host, making this process an excellent drug target. In order to attempt to control, or better yet, eradicate this pathogen, a greater understanding of its entry mechanism, a fundamental aspect of the viral life cycle, is required. It is well established that Influenza A virus (IAV) fusion is driven by the viral glycoprotein hemagglutinin (HA), which when exposed to low pH transitions from a meta-stable pre-fusion to post-fusion state. However, it is becoming increasingly apparent that the ionic balance of the endosomes also has a significant role in the entry of enveloped viruses. Specifically, our preliminary studies on Influenza virus suggest that K+ concentrations within the endocytic pathway play a significant role in Influenza A fusion events. Through the use of broad-spectrum K+ channel inhibitors, a dose-dependant reduction in Influenza infectivity in tissue culture can be observed. Further to these studies, we have isolated Affimers (novel antibody-like proteins that can be produced in large quantities in E. coli) that recognise HAs from different Influenza subtypes (H3N2 and H1N1). Using these Affimers, we aim to develop an early detection method to distinguish between bacterial respiratory infections and ‘the flu’ to alleviate mounting pressures on an already diminishing antibiotic treatment system.
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Effect of antibacterial lipids on biofilm formation by Streptococcus mutans
More LessStreptococcus mutans is the major cariogenic organism associated with Dental Caries, a widespread chronic disease of the oral cavity (Muras et al. 2018). It is associated with oral biofilm formation, production of organic acids, and has the capacity to out-compete non-cariogenic commensal species (Lemos et al. 2013). Recent studies carried out on fatty acids demonstrated effective antimicrobial activity against S. mutans (Hughes, 2014). This study will evaluate the activity of lipids on S. mutans biofilm formation and proposes that dietary constituents may be used as a natural therapy to maintain oral hygiene. Clinical isolate S. mutans 3014 D5929 was exposed to various concentrations of lipid for 24 h. Crystal violet assay was performed for quantification of biofilm biomass. Fluorescent microscopy using SYTO® 9 and Alexa Fluor® 647-labelled dextran conjugate was performed to visualise biofilm formation pre- and post-exposure to MCO. Biofilm biomass was reduced for all lipid concentrations. Fluorescent microscopy indicated a significant reduction in bacterial cell number and a lack of structural biofilm upon exposure to fatty acid mixtures when compared to control. The research demonstrated that lipid does have S. mutans based antimicrobial and antibiofilm capabilities.
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D-Amino acids do no inhibit biofilm formation in Staphylococcus sp.
More LessD-amino acids are responsible for cell wall re-modelling in Staphylococcus and are capable of inhibition and mature biofilm disassembly. Staphylococcus aureus and Staphylococcus epidermidis are recognised as recurrent nosocomial pathogens and a common cause of biofilm-associated infections. The combination of amino acids used in the study consisted of d- and l- isomers of tyrosine, methionine, tryptophan and phenylalanine. A semiquantitative microplate crystal violet assay was used to assess the effect of amino acids on biofilm development. Biofilm viability staining using fluorescent microscopy was performed to assess the effect of the amino acid mixtures on biofilm development on submerged surfaces. None of the amino acids when tested individually or as a mixture could reduce biofilm formation. However, at the highest concentration tested 25 mmol 1−1 equimolar D-amino acid mixture of tryptophan, phenylalanine, tyrosine and methionine caused a considerable biofilm inhibition in three Staphylococcus strains. Microscopy analysis showed that initial surface attachment remained unaffected at 25 mmol 1−1 mixture of d-amino acids but bacteria did not proceed to form mature biofilms. This suggests inhibition of protein synthesis or a lack of polysaccharide extracellular adhesin formation as no aggregates were observed. The reported bioactivity of D-amino acid on biofilm development and disassembly has been conflictual. It has been established that D-amino acids are incorporated in the bacterial cell wall suggesting they play a role in the complexity of biofilm lifecycle. However, our study indicates that they play no direct role in the inhibition of biofilm formation in Staphylococcus.
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