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Volume 1,
Issue 1A,
2019
Volume 1, Issue 1A, 2019
- Poster Presentation
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- Virology Workshop: Clinical Virology
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Retrospective point-prevalence study of Enterovirus D68 (EV-D68) detection in the symptomatic paediatric population presenting to the Children’s Hospital Emergency Department, RBHSC, Northern Ireland
More LessEnterovirus D68 (EV-D68) detection has recently been associated with severe neurological symptoms in adults and children in European countries. The spectrum of disease by EV-D68 ranges from asymptomatic to acute respiratory symptoms, hospitalisation, and sporadically to neurological symptoms, including acute flaccid paralysis (AFP) and acute flaccid myelitis (AFM), and death. This has led to increased vigilance for detection of enterovirus D68, especially in cases that present with the more severe clinical syndromes. In normal cases, enterovirus is transmitted by faecal-oral routes and/or respiratory routes, however in the case of EV-D68 it is almost exclusively passed through respiratory transmission. An infection of EV-D68 is generally distinguished by its rapid onset of disease. The incubation period of the disease between 3 to 5 days, which is unlike the other enteroviruses which usually have an incubation period of around 10 days. This study describes a retrospective analysis of 150 non-invasive respiratory specimens (nose/throat or throat swabs) collected from paediatric outpatients presenting to Childrens Hospital Emergency Department with respiratory and/or CNS symptoms including fever and seizure. Specimens have been collected from November 2017- November 2018. All specimens tested by four RT-PCR assays for Pan-Enterovirus, EV-D68 specific, human Rhinovirus and RNaseP (quality control). Results on prevalence of EV-D68, as single or co-infection, are presented. Results indicate low prevalence of EV-D68 in the Northern Ireland symptomatic paediatric population. As EV-D68 is an emerging infection it is critical to remain vigilant particularly in the case of neurological presentation.
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Lyme disease in Northern Ireland – a look back on serological testing
More LessBackgroundLyme 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.
MethodsWe 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.
ResultsOf 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.
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Validation of a hepatitis B virus (HBV) Next Generation Sequencing (NGS) pipeline at Barts Health NHS Trust
More LessAt 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.
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Investigating the impact of influenza point-of-care testing – what are the benefits?
More LessPoint-of-care testing (POCT) for influenza viruses is being used increasingly in NHS hospitals. In the 2017/18 influenza season, NHS Greater Glasgow and Clyde implemented the Cepheid GeneXpert System for the detection of influenza A, B and respiratory syncytial virus in 7 wards across 5 hospital sites. To evaluate the impact of influenza POCT in Glasgow Royal Infirmary during the 2017/18 influenza season, we retrospectively compared data from 150 influenza A infected patients. We analysed data from 100 patients who were diagnosed using a laboratory based in-house respiratory PCR assay (50 patients from 2016/17 and 50 patients from the 2017/18 season) and 50 patients who were diagnosed using POCT in the 2017/18 season. The aim of the study was to investigate whether POCT impacted patient management and outcome. General linear models were used to test for an association between POCT and a number of outcomes, whilst accounting for host factors. These outcomes included: (1) time from review to influenza result (2) admission status (3) length of hospital stay (4) patient outcome (5) duration of antibiotics (6) antiviral treatment (7) patient isolation and (8) the number of duplicate influenza tests performed. In addition to investigating the impact of POCT, we used this opportunity to explore additional factors that may affect the outcomes of patients infected with influenza A, including: age, sex, imaging results and underlying risk factors.
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- Virology Workshop: Gene Expression and Replication
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Investigating alternative AUG usage in avian Influenza A virus segment 2
More LessInfluenza A viruses (IAV) have a segmented, negative sense RNA genome. PB1-F2 is an IAV accessory protein encoded by segment 2, in the +1 reading frame. IAVs from avian hosts generally encode full length PB1-F2s, which contrasts with human IAVs which frequently have C-terminal truncations. Many reported activities of PB1-F2, including innate immune antagonism, require motifs in its C-terminal domain. Full length PB1-F2 is translated from AUG 4 of segment two, but one or more of AUGs 7, 8 and 9 may also serve as independent initiation codons for the C-terminal domain. Products from the AUGs 7-9 are expressed during infection by a vaccine strain IAV, but their presence or absence had no effect on virus growth in vitro. We generated a panel of isogenic viruses, containing segment 2 from an avian H5N1 IAV, which differed in the presence or absence of the various AUG start codons in segment 2. No difference in growth kinetics in vitro or viral polymerase activity, measured using a mini-replicon assay, was observed for any of these mutants. However a significant difference in mean plaque size on MDCK cells was seen when individual changes were made to any of AUGs 7-9, suggesting a subtle effect on virus fitness possibly caused by loss of expression of PB1-F2 C-terminal fragments. In addition structural predictions suggest that the AUG mutations will affect secondary structure of full length PB1-F2. Our works suggests segment 2 protein expression from multiple AUGs could impact of the virus replication cycle.
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Antagonism of mosquito innate immunity by the chikungunya virus nsP3 protein
More LessChikungunya virus (CHIKV) is a re-emerging Alphavirus transmitted by Aedes mosquitos and causing fever, rash and chronic arthralgia. There are no vaccines or antiviral agents available for CHIKV therefore it is important to understand the molecular details of virus replication. To address this, we previously conducted a mutagenic analysis of the central alphavirus unique domain (AUD) of the CHIKV non-structural protein 3 (nsP3), testing replication of a subgenomic replicon in a variety of mammalian and mosquito (Aedes albopictus) cell lines. One mutant (M219A) exhibited a different phenotype in two Aedes albopictus cell lines (U4.4 and C6/36): replicating as wildtype in C6/36 but was blocked in U4.4. As U4.4 cells have an intact RNAi response whereas C6/36 have a frameshift mutation in the Dicer-2 (Dcr2) gene and express an inactive Dcr2 protein, we proposed that the replication of M219A was suppressed by the RNAi antiviral response in U4.4 cells, while wild type nsP3 was able to counteract this response. To further investigate this hypothesis we have extended the mutagenic analysis to screen other residues in proximity with M219 within the AUD. All of these mutants retain wildtype levels of replication in mammalian and C6/36 cells, except for W220A which replicates poorly. Evaluation of the replication of these mutants in U4.4 is ongoing. We are also pursuing a CRISPR/Cas9 approach to ablate expression of Dcr2 in U4.4 cells, and generating stable C6/36 cells expressing Dcr2 to confirm our hypothesis. Our studies will shed light on how CHIKV nsP3 can antagonise mosquito innate immunity.
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Analysis of the novel role of NS5A domain I in the assembly of infectious hepatitis C virus particles
More LessHepatitis C virus (HCV) is an enveloped virus with a positive-sense, single-stranded RNA of approximately 9.6 kb, a member of the genus Hepaci virus within the family Flaviviridae. The genome contains a single large open reading frame encoding a 3000 residue polyprotein. The non-structural 5A protein (NS5A) is a highly phosphorylated protein, whichis comprised of three domains (I, II and III). Previously, we demonstrated that two residues within NS5A domain I (V67 and P145) play critical roles in HCV assembly challenging the dogma that NS5A domain I exclusively participated in genome replication. In this study, we identified 8 surface exposed residues of domain Iwhich were located in close proximity to V67 and P145. The mutants were cloned into a JFH-1 derived subgenomic replicons (mSGR-luc-JFH1) to confirm whether they are required in genome replication. The results of luciferase assay suggested that I52A exhibited the same phenotype as V67A and P145A and is a further candidate for regulating assembly of HCV. In parallel we sought to investigate whether domain I was involved in the interaction of NS5A with cyclophilin A (CypA), a cellular peptidyl-prolyl isomerase required for HCV replication. CypA can be inhibited by cyclosporin A (CsA), which also inhibits HCV genome replication. Surprisingly, all three mutant replicons (I52A, V67A and P145A) were more sensitive to CsA treatment than wildtype, suggesting that domain I does indeed interact with CypA. Ongoing studies will therefore investigate the roles of both domain I and CypA in genome replication and assembly of infectious HCV particles.
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The modulation of autophagy by African swine fever virus
More LessAfrican swine fever virus (ASFV) causes a lethal haemorrhagic disease of domestic pigs with mortality rates of up to 100 %. An outbreak in Russia in 2007 has since spread into Europe. There is currently no vaccine available, however infection with attenuated strains of ASFV can protect against infection with closely related virulent strains. Autophagy is a conserved, essential cell process that regulates multiple pathways that are critical for mounting an effective immune response. Experiments have shown that inhibiting the ability of viruses to regulate autophagy can lead to enhanced immune responses. We have shown that ASFV does not require autophagy for replication and that autophagosome formation is inhibited during infection. In addition, through analysis of key proteins in the upstream autophagy pathway, we describe a novel mechanism of ASFV inhibition of autophagy. This research will expand our understanding of the interaction between ASFV and the autophagy pathway with the potential that a low virulent ASFV strain with an altered ability to modulate autophagy will provide enhanced immunity against virulent isolates.
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Ribosome profiling of porcine reproductive and respiratory syndrome virus
Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus of huge economic importance, infecting the porcine host leading to infertility, morbidity, and mortality. Its genome contains a canonical −1 programmed ribosomal frameshift (PRF) site that facilitates expression of the viral replicase, and a second, non-canonical signal which induces both −1 and −2 PRF to generate alternative forms of a viral non-structural polypeptide, nsp2. In contrast to canonical frameshift sites, the frameshift at the non-canonical site is not stimulated by a downstream secondary RNA structure, but is instead the first known example of protein-directed frameshifting, stimulated by a trans-acting complex of a viral (nsp1β) and a cellular (PCBP) protein. We investigated frameshifting in PRRSV by ribosome profiling, using the vaccine strain, SD95-21, and a derivative with mutations at the nsp2 site that render it PRF-defective. Highly efficient PRF was observed at both the canonical, RNA pseudoknot-dependent −1 PRF site (efficiency of43–56 %), and the nsp1β/PCBP-dependent site (combined −1 and −2 PRF efficiency of 20–24 %). Investigations are underway as to whether the presence of nsp1βduring viral infection stimulates non-canonical frameshifting on host mRNAs. We also carried out RNA-Seq and differential transcription analysis in parallel with ribosome profiling to garner further insight into the functions of thensp2transframe proteins, previously shown to be involved in innate immune suppression. This ribosome profiling analysis has also revealed the presence of a short but highly expressed upstream ORF in the 5’UTR of PRRSV.
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Functional RNA structures as targets in emerging arboviruses
More LessEmerging arthropod-borne viruses, such as Zika virus (ZIKV) and Chikungunya virus (CHIKV), represent a significant and increasing threat to global health. Despite the expanding prevalence of their vectors, the Aedes sp.mosquitoes, and the potential for major epidemics, there are currently no specific antiviral compounds or vaccines available for either viral pathogen. The positive-strand genomes of ZIKV and CHIKV, members of the flavivirus and alphavirus genera respectively, contain functional, structured cis-acting RNA elements. We are investigating a range of approaches for targeting these RNA elements and analysing the effect on virus replication at different stages of their life cycle. Gaining high resolution structural data is essential prior to targeting RNA elements, and consequently, we mapped RNA structural elements within the ZIKV 5’ genome region using a combination of biochemical SHAPE probing, thermodynamic predictions and phylogenetic analysis. We are currently validating our structural data by analysis of mutant phenotypes in a reverse genetic system. Using antisense locked nucleic acid oligonucleotides (antisense-LNA), we demonstrated that functional RNA elements in CHIKV can be specifically targeted – inhibiting replication in both sub-genomic replicon and infectious virus systems. Surface plasmon resonance confirmed that the antisense-LNA binds to a specific stem-loop target with aKd of 310 nM and has an IC50 of 35 nM in the sub-genomic replicon system. In future work, we aim to investigate selection of RNA-aptamers against CHIKV and target ZIKV genomic stem-loops using antisense-LNAs.
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Functional and structural studies of Chikungunya Virus nsP1 Protein
More LessChikungunya virus (CHIKV) is a single-stranded, positive-sense RNA virus of the Alphavirus genus. CHIKV is an arbovirus, whose spread is mediated by Aedes species mosquitos and is associated with debilitating joint pain and febrile symptoms in infected humans. A lack of vaccine or specific antivirals, combined with increasing global spread, has facilitated the re-emergence of CHIKV in recent years. Our research focuses on the CHIKV encoded non-structural protein-1 (nsP1), which has methyltransferase activity and is essential for virus genome replication. Through a combination of structural, biochemical and reverse genetic approaches, we aim to investigate the relationship between the molecular structure and both canonical and non-canonical functions of nsP1, at different stages of CHIKV replication. We demonstrated that substitution of an in-frame methionine (M24), towards the N-terminal of nsP1, severely inhibits CHIKV replication in a host cell-dependent manner. Specifically, we demonstrated that an M24>A substitution had no significant effect on sub-genomic replicon replication but blocks production of infectious CHIKV virions – suggesting a role in later stages of virus replication, such as packaging or egress, rather than genome replication or translation. Utilising such a reverse genetic approach, analysis of a panel of M24 substitutions has improved our understanding on non-canonical yet essential functions of nsP1 during CHIKV replication. In order to further elucidate both the structural and biochemical basis for the observed mutant phenotypes, we developed a system for bacterial expression and purification of recombinant CHIKV nsP1 and have established crystallography trails, prior to analysis of the molecular structure by X-ray crystallography.
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Structural and phenotypic analysis of Chikungunya Virus RNA structures during viral genome replication and translation
Chikungunya virus (CHIKV) is a pathogenic, positive-sense RNA virus of the Alphavirus genus, which causes fever and debilitating joint pain in humans. CHIKV is transmitted by Aedes mosquitoes and is currently re-emerging in the absence of approved vaccines or antiviral therapies. One approach to vaccine production is to attenuate CHIKV replication through disruption of genomic RNA secondary structures. We have mapped the RNA structure of the 5’ region of the CHIKV genome using selective 2’-hydroxyl acylation analysed by primer extension (SHAPE) to investigate intramolecular base-pairing at single-nucleotide resolution. We have identified five highly-conserved RNA structures within the nsP1-coding region of ORF-1 and, using a reverse genetics approach, determined their impact on virus replication in infectious virus and luciferase-reporter subgenomic replicon systems. Our results suggest that RNA structures within the nsP1-coding region are required for efficient CHIKV genome replication in mammalian and mosquito cells, potentially via vertebrate/invertebrate host-specific mechanisms. For example, disruption of one of the stem-loops inhibits CHIKV replication in mammalian cell lines, while having no significant effect in mosquito cells. Restoration of the structure via compensatory silent mutations restores replication – indicating RNA structure-dependent enhancement of CHIKV replication. Conversely, disruption of an adjacent stem-loop inhibits CHIKV replication in mosquito cells but not in mammalian cell lines. Our structural data also suggests that higher-order interactions within this region impact CHIKV replication. As arboviruses continue to re-emerge, it is critical that we improve our understanding of their replication cycles. RNA structures may constitute novel targets for vaccine attenuation or drug design.
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Application of next generation sequencing for the elucidation of genes and pathways involved in the host response to bovine respiratory syncytial virus
High rates of calf mortality in the first 12 months of life, results in significant economic losses in Europe and the USA. Bovine respiratory disease (BRD) accounts for the largest proportion of calf mortality. There is a paucity of literature concerning the host response to BRD. In a controlled challenge study in artificially reared dairy calves [155 (S.D. 14) kg], the influence of the host response to bovine respiratory syncytial virus (BRSV) was examined. At AFBI Holstein-Friesian calves were either challenged with BRSV (n=12) or mock challenged with phosphate buffer saline (n=6). Calves were euthanised on day 7 post-challenge. Bronchial lymph nodes were collected and flash-frozen at −80 °C. RNA was extracted and sent to the University of Missouri’s DNA Core Facility for RNA-Seq library preparation and sequencing. Sequenced reads were adapter trimmed, quality assessed using FastQC and aligned to the bovine genome (UMD 3.1) using STAR. Differential gene expression analysis was performed using EdgeR, and pathway and gene ontology analyses were carried out using g:Profiler and Ingenuity Pathway Analysis (IPA). There was a clear separation between BRSV challenged and control calves based on log2 fold gene expression changes, despite an observed mild clinical manifestation of the disease. There were 934 differentially expressed genes (DEG) (P<0.05, FDR<0.1, fold change >2) between the BRSV challenged and control calves. Over-represented pathways and gene ontology terms among the DEG were associated with immune responses and included: GO:0051607 defense response to virus, the KEGG pathway Influenza A and the IPA pathway Interferon Signaling.
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Generation of a recombinant GFP-tagged infectious bronchitis virus (IBV)
More LessInfectious Bronchitis Virus (IBV) is a highly contagious gammacoronavirus which infects poultry. Using reverse genetics, enhanced green fluorescence protein (eGFP) has been inserted into a pathogenic strain of IBV, M41, in place of a newly identified open reading frame (ORF), ORF7. ORF7 has been identified in several IBV strains including M41 and apathogenic strain Beau-R. ORF7 is located immediately downstream of the N gene, preceding the 3’ UTR. This region has been chosen because we are interested in investigating whether a protein can be transcribed from the associated transcription regulatory sequence (TRS-B). M41 has a deletion at the 3’ end of the genome and does not encode all of ORF7, suggesting it is not essential for the viral replication and is not required for a pathogenic phenotype. The TRS-B, TAACA for ORF7 in Beau-R, is non-canonical and is located after the stop codon for the N gene. In M41 only part of the TRS-B is present, TAA, so nucleotides CA were added alongside the eGFP sequence. A number of viruses were successfully rescued. Growth kinetics in primary Chicken Kidney cells (CK) were comparable to the parent virus M41-K. The stability of the eGFP sequence has been assessed in CK cells and in embryonated eggs. The newly constructed TRS-B however was not utilised suggesting that it is not solely the TRS-B that controls the transcription of ORF 7.
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Visualising influenza virus replication using a click chemistry approach
More LessInfluenza A viruses (IAVs) have a significant impact on public health through seasonal epidemics. Moreover, the sporadic emergence of zoonotic and pandemic strains represents an additional global threat. Understanding how novel IAV strains adapt to infect and transmit between humans is essential for effective surveillance and may provide rationale for anti-viral therapeutics. IAVs replicate their RNA genomes in the host cell nucleus. Host proteins, such as the proviral factor ANP32, are co-opted to support viral replication and transcription, whilst other inhibitory factors act to restrict. Ultimately the outcome of infection in different species depends on compatibility with these factors, however the precise nuclear localisation of these interactions is not clearly defined. It is not known whether viral RNA synthesis takes place at discrete sites, or whether the virus modifies the cell to produce viral factories in a similar manner to other viruses. We are utilising a click chemistry approach to investigate the spatial details of IAV replication and to probe the differences in nuclear localisation between human-adapted vs poorly-adapted avian origin IAVs, to gain insight into mechanisms of host restriction. Influenza genomes labelled with the click reagent 5-ethynyl uridine (5-EU) remain infectious and can be visualised through the cycloaddition of azide-tagged fluorescent dyes. Utilising this tool, the co-localisation of incoming viral particles with nuclear sub-domains, as well as key host factors, are being examined. Moreover, the localisation of human- vs avian-adapted virus in human and avian cells, as well as cell lines lacking key host factors, are being compared.
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HIV-1 infectivity of cells is enhanced at mitosis: a role for Vpr?
More LessRestriction 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).
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- Virology Workshop: Innate Immunity
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Understanding virus resistance due to ISG15-loss-of-function
Viral infections induce profound cellular responses resulting the expression of hundreds of IFN-stimulated genes (ISGs). Some ISGs have specific antiviral activity, while others regulate the cellular response. For most viruses, the specific antiviral ISG(s) is not known, which has potential consequences for the quest for new therapeutics. The ubiquitin-like protein ISG15 is a major regulator of antiviral response and inherited ISG15-deficiency leads to autoinflammatory interferonopathies where patients exhibit elevated ISG expression in the absence of infection. Using CRISPR/Cas9 knockout technology, we have recapitulated these effects in cultured cells, confirming ‘free’ ISG15’s role as a central regulator of type-I IFN antiviral response. We also show that during an antiviral response, ISG15-deficiency leads to significant physiological defects (inhibition of translation and proliferation) and resistance to parainfluenza viruses. We asked if virus resistance was due to the direct antiviral activity of ISGs, or whether cells were non-permissive due to physiological defects. We took advantage of the knowledge that IFIT1 is the principle antiviral ISG for parainfluenza virus 5 (PIV5). Knockdown of IFIT1 restored PIV5 infection in ISG15-deficient cells, confirming that resistance was due to the antiviral response and not due to physiological state related to ISG15-deficiency. We also compared infections with related viruses where IFIT1 has known intermediate antiviral activity (PIV2) and low activity (PIV3); restoration of replication with these viruses reflected their sensitivity to IFIT1 restriction. Based on the observations in IFIT1-knockdown cells, we propose a novel platform for the identification of antiviral ISGs based on recovery of virus infection.
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The VP4 protein from a very virulent IBDV strain antagonises type I IFN responses to a greater extent than the VP4 from a classical strain
More LessInfectious bursal disease virus (IBDV) infects B cells in the bursa of Fabricius (BF) causing morbidity, mortality and immunosuppression in infected chickens. Classical strains (e.g. F52/70) cause 1–2 % mortality whereas very virulent strains (e.g. UK661) cause over 60 % mortality for reasons that remain poorly understood. We inoculated birds with either F52/70 or UK661, and found that the expression of pro-inflammatory and type I IFN-related genes was significantly down-regulated in UK661 compared to F52/70 infected birds (P<0.05), despite no statistically significant difference in peak virus titres between the two strains. This was also observed in vitro in an immortalised B cell line where UK661 caused significantly reduced IFNβ and Mx1 expression compared to F52/70 (P<0.05). The IBDV protease (VP4) has previously been reported to act as a type I IFN antagonist, although it remains unknown whether this is characteristic of all IBDVs or a strain-specific phenomenon. Using a luciferase reporter assay, we compared the IFNβ production in DF-1 cells in response to poly I:C stimulation in the presence of eGFP-VP4 expression plasmids, finding UK661 VP4 was able to down-regulate IFNβ production to a greater extent than F52/70 VP4 (P<0.01). There are 9 amino acid differences between the two VP4 proteins and we are identifying those contributing to the observed phenotype. Taken together, our data suggest that the VP4 protein in very virulent IBDV strains evolved a greater ability to antagonise type I IFN responses than classical strains which may, in part, explain their enhanced virulence.
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Identification of host proteins that interact with non-structural proteins-1α and -1β of PRRSV-1
More LessPorcine reproductive and respiratory syndrome viruses (PRRSV) are responsible for the most important infectious disease affecting the global pig industry, causing respiratory disease in piglets and reproductive failure in sows. Both species of PRRSV (PRRSV-1 and −2) are rapidly evolving and existing vaccines are failing to control the PRRS panzootic. PRRSV produces 16 non-structural proteins (NSPs) that are involved in viral replication and/or modulating the host immune response. Previous studies have shown that PRRSV NSP1α and NSP1β modulate host cell responses; however, the underlying molecular mechanisms remain to be fully elucidated. PRRSV-1 strains predominate in Europe but have been studied far less than PRRSV-2, therefore, this project aims to identify and characterise novel PRRSV-1 NSP1-host protein interactions. NSP1α and NSP1β from a representative PRRSV-1 subtype 1 strain (215-06) were screened using the yeast-2-hybrid (y-2-h) system and a cDNA library generated from porcine alveolar macrophages – the primary target cell of PRRSV-1. The screens identified 62 and 127 putative binding partners for NSP1α and NSP1β, respectively. Potential binding partners involved in IFN signalling, the NF-κB pathway, ubiquitination and nuclear transport have been selected for confirmation and characterisation. Identifying and characterising these novel interactions will increase our understanding of how PRRSV-1 NSP1α/β modulates the host cellular immune response, which could subsequently be exploited to rationally attenuate PRRSV-1 as a basis for improved vaccines.
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Study of the regulation of human IFIT1 stability
More LessInterferon-induced proteins with tetratricopeptide repeats (IFITs) are produced in both interferon-dependent or interferon-independent manners after pathogen-associated molecular pattern recognition. Four IFITs (IFIT1, IFIT2, IFIT3 and IFIT5) have been characterized in humans. They are cytoplasmic proteins with repetitive tetratricopeptide repeats, protein motifs well characterized to mediate protein-protein interactions. IFITs play several functions in cells and their antiviral roles are well established. IFIT1 principally binds non-self cap0 mRNAs and inhibit their translation. On its own, IFIT1 overexpresses poorly in cells. Co-expression with IFIT3 enhances IFIT1 protein levels. Likewise, downregulation of endogenous IFIT3 decreases the expression of IFIT1 at the protein level. The stabilization is dependent on the interaction of the C terminus of IFIT3 with IFIT1 via a specific motif in both proteins, disruption of which greatly reduced IFIT1 stability. It is currently unclear why efficient IFIT1 expression is regulated by its interaction with IFIT3 in this manner. To address this, we have begun to investigate the process of IFIT1 degradation. We have used a range of inhibitors to disrupt specific cellular protein degradation pathways. Surprisingly, inhibition of proteasome and lysosome pathways showed protection of IFIT1 from active degradation, suggesting the protein may be degraded via various routes. We will discuss our current efforts to generate stabilized IFIT1 mutants using random and insertional mutagenesis to identify motifs that may contribute to IFIT1 turnover. These findings will contribute to a deeper understanding of IFITs role during the immune response and may identify methods by which their function can be manipulated.
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Prevalence and resistance pattern of uropathogens from community settings of different regions: an experience from India
Sarita Mohapatra, Rajashree Panigrahy, Vibhor Tak, Shwetha J. V., Sneha K. C., Susmita Chaudhuri, Swati Pundir, Deepak Kocher, Hitender Gautam, Seema Sood, Bimal Kumar Das, Arti Kapil, Pankaj Hari, Arvind Kumar, Rajesh Kumari, Mani Kalaivani, Ambica R., Harshal Ramesh Salve, Sumit Malhotra and Shashi Kant
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