المؤلفون: Steve Goodbourn, Munir Iqbal, Wendy S. Barclay, Holly Shelton, Craig Ross, Joe James, Nikki Smith, Paul Digard

المصدر: James, J, Smith, N, Ross, C, Iqbal, M, Goodbourn, S, Digard, P, Barclay, W S & Shelton, H 2019, ' The cellular localization of avian influenza virus PB1-F2 protein alters the magnitude of IFN2 promoter and NFκB-dependent promoter antagonism in chicken cells ', Journal of General Virology . https://doi.org/10.1099/jgv.0.001220Test

مصطلحات موضوعية: 0301 basic medicine, animal structures, viruses, 030106 microbiology, Chicken Cells, Biology, medicine.disease_cause, Virus, Viral Proteins, 03 medical and health sciences, Virology, Influenza A Virus, H9N2 Subtype, Influenza A virus, medicine, Animals, Promoter Regions, Genetic, Gene, Poultry Diseases, Cellular localization, Influenza A Virus, H5N1 Subtype, NF-kappa B, virus diseases, Interferon-beta, biochemical phenomena, metabolism, and nutrition, In vitro, Cell biology, 030104 developmental biology, Cytoplasm, Influenza in Birds, Antagonism, Chickens

الوصف: The accessory protein, PB1-F2, of influenza A virus (IAV) functions in a chicken host to prolong infectious virus shedding and thus the transmission window. Here we show that this delay in virus clearance by PB1-F2 in chickens is accompanied by reduced transcript levels of type 1 interferon (IFN)-induced genes and NFκB-activated pro-inflammation cytokines. In vitro, two avian influenza isolate-derived PB1-F2 proteins, H9N2 UDL01 and H5N1 5092, exhibited the same antagonism of the IFN and pro-inflammation induction pathways seen in vivo, but to different extents. The two PB1-F2 proteins had different cellular localization in chicken cells, with H5N1 5092 being predominantly mitochondrial-associated and H9N2 UDL being cytoplasmic but not mitochondrial-localized. We hypothesized that PB1-F2 localization might influence the functionality of the protein during infection and that the protein sequence could alter cellular localization. We demonstrated that the sequence of the C-terminus of PB1-F2 determined cytoplasmic localization in chicken cells and this was linked with protein instability. Mitochondrial localization of PB1-F2 resulted in reduced antagonism of an NFκB-dependent promoter. In parallel, mitochondrial localization of PB1-F2 increased the potency of chicken IFN 2 induction antagonism. We suggest that mitochondrial localization of PB1-F2 restricts interaction with cytoplasmic-located IKKβ, reducing NFκB-responsive promoter antagonism, but enhances antagonism of the IFN2 promoter through interaction with the mitochondrial adaptor MAVS. Our study highlights the differential mechanisms by which IAV PB1-F2 protein can dampen the avian host innate signalling response.

وصف الملف: application/pdf

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::de9042f2c81408feccaebb57f5e61d68Test
https://doi.org/10.1099/jgv.0.001220Test

المؤلفون: Eleanor Gaunt, Rute Maria Pinto, Ola Lee, Paul Digard, Hui Min Lee

المصدر: Access Microbiology. 2

مصطلحات موضوعية: General Materials Science, Computational biology, Biology, Reverse genetics, Coding (social sciences)

الوصف: Bovine rotavirus (RV) infection causes severe diarrhoea in young dairy calves and has a significant economic impact on livestock production as a result of high morbidity and mortality caused. Development of technologies to engineer infectious RV using an entirely plasmid-based reverse genetics (RG) system has proven challenging. A breakthrough was made when Kanaiand co-authors (PNAS, 2017)developed a plasmid-only-based RG system for the simian RV strain SA11.We are currently developing an analogous RG system for the bovine RF RV strain. Having parallel systems for different RV strains will help to validate phenotypic changes induced by site-directed mutagenesis (SDM) within the RV genome. The coding capacity of the 11-segmented dsRNA RV genome has been largely unexplored. Using bioinformatic analyses, we have identified four segments with up to five putative alternative initiation codons which are in moderate or strong Kozak context. Furthermore, some occur in segments for which the canonical start codon occurs within 15 nucleotides of the start of the segment, further suggesting the possibility of alternative translation start sites to generate coding diversity. We are now applying our RG systems to investigate RV coding capacity using TnT transcription assays, radiolabelling and SDM.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::44813771550471e4fe5fd9d9ef065d0aTest
https://doi.org/10.1099/acmi.ac2020.po0558Test

المؤلفون: Wioleta Drobik-Czwarno, Jacqueline Smith, Paul Digard, Anna Wolc, Janet E. Fulton, Cal Donnelly

المصدر: Access Microbiology. 2

مصطلحات موضوعية: Whole genome sequencing, Genetics, Candidate gene, Genome editing, medicine, General Materials Science, Single-nucleotide polymorphism, Genome-wide association study, Biology, medicine.disease_cause, Gene, Influenza A virus subtype H5N1, Genetic association

الوصف: DNA samples collected from survivors of recent outbreaks of highly pathogenic avian influenza (HPAI) in Mexico and the USA have provided a rare opportunity to study the genetic mechanisms underpinning susceptibility of chickens to this devastating and economically impactful disease which normally exhibits 70-100% mortality in the chicken host. Whole Genome Sequence (WGS) data has been used to perform Genome-Wide Association Studies (GWAS), which have highlighted single nucleotide polymorphisms (SNPs) segregating significantly between survivors and controls, with a pedigreed experimental group exhibiting a highly significant signal on chicken chromosome 2 in the region of a biologically relevant gene. Candidate SNPs for resilience are currently being validated using in vitro gene editing methodologiesthat modulate candidate gene expression to investigate the effect on viral replication and cellular response to HPAI infection. A detailed understanding of the genomic resilience to HPAI from this study will have implications for both the poultry industry and for public health.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::8b84a7ad55c3160dca8a151220b6ecf6Test
https://doi.org/10.1099/acmi.mim2019.po0018Test

المؤلفون: Finn Grey, Samantha J. Griffiths, Nhan T. Pham, Paul Digard, Chen-Hsuin Lee, Manfred Auer, Juergen Haas

المصدر: Access Microbiology. 1

مصطلحات موضوعية: Human cytomegalovirus, Reporter gene, Small interfering RNA, viruses, Protein degradation, Biology, medicine.disease, Virus, Cell biology, Viral replication, Transcriptional regulation, medicine, General Materials Science, Gene

الوصف: As an obligate intracellular parasite, human cytomegalovirus (HCMV) completely relies on host machinery to replicate. Understanding which host factors are required for virus replication contributes to our understanding of virus biology and cell biology, identification of potential targets for antiviral therapy. High-throughput small interfering RNA (siRNA) screens are a powerful approach to identify novel host-virus interactions. Conventional screens often use reporter genes as a proxy for virus replication, rather than measuring production of infectious virus. We developed a two-step siRNA screen that independently measured primary replication and virus production. Screening with a library targeting almost 7000 genes, we identified 37 genes involved in early stages of HCMV replication and 15 genes specifically involved in later aspects, such as late gene expression, assembly and egress. These include factors in ubiquitin-dependent protein degradation pathway, and components of the mediator complex. Furthermore, we showed that the induction of SIN3A, a transcriptional regulator that forms a repressor complex with histone deacetylase 1 and 2, is essential for late gene expression and virus production. This study demonstrates a powerful two-step high throughput approach which identifies key host factors underpinning HCMV replication and informs our understanding of how the virus interacts with its host.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::8c6edaf9adbd4c677076628d08ad7e91Test
https://doi.org/10.1099/acmi.ac2019.po0068Test

المؤلفون: Elizabeth Billington, Rute Maria Pinto, Paul Digard, Holly Shelton

المصدر: Access Microbiology. 1

مصطلحات موضوعية: biology, viruses, Mutant, medicine.disease_cause, Virology, Genome, Influenza A virus subtype H5N1, Virus, Avian Influenza A Virus, Start codon, Viral replication, medicine, biology.protein, General Materials Science, Polymerase

الوصف: Influenza 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.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::8ed556ea3deb5379ca8ebbf329d5b1f4Test
https://doi.org/10.1099/acmi.ac2019.po0071Test

المؤلفون: Deimante Lukosaityte, Jean-Remy Sadeyen, Munir Iqbal, Paul Digard

المصدر: Access Microbiology. 1

مصطلحات موضوعية: medicine.drug_class, Biology, medicine.disease_cause, Monoclonal antibody, Virology, Fragment crystallizable region, Virus, Neutralization, Antigen, Influenza A virus, medicine, biology.protein, Single-chain variable fragment, General Materials Science, Antibody

الوصف: Monoclonal antibodies (mAbs) that neutralize influenza A virus have been shown to be potent therapeutic reagents if used pre- or post – exposure to the pathogen. Whilst the majority of this research has focused on human antibody therapeutics, there is an urgent need for generation of antibodies able to neutralize avian influenza viruses (AIV). Several mAbs against H9N2 virus have previously been generated using mouse hybridomas. To facilitate development of passive immunization strategies, the variable chains of these antibodies were characterized in the context of isotype and species specific Fc fragments. Replacement of the IgG2 Fc region with an IgG1 Fc region enhanced neutralizing activity for one of the tested antibodies. Additionally, chicken chimeric antibodies were generated which showed comparable neutralization titres to those generated by the original hybridomas. To identify if antibodies could function as a single chain variable fragment antibodies (scFvs) these were produced in insect S2 cells. This confirmed that H9N2 virus can be neutralized by scFvs. However, an example with high HI titres but lost detectable neutralizing activity was found, possibly due to the loss of bivalent interaction between antigen and antibody. Antibodies showing superior neutralization activity in vitro will be subsequently tested for their potency in in vivo infection. We propose that passive immunization can reduce the impact of AIV in poultry by inducing immediate protection and bypassing immunocompromised individuals.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::8b28e4224a326a7fd734d07066db76ddTest
https://doi.org/10.1099/acmi.ac2019.po0194Test

المؤلفون: Rachael S. Wash, David Goulding, Stacey Efstathiou, E-Pien Tan, Sabrina Calabressi, Jürgen Haas, Paul Digard, Samantha J. Griffiths, Helen M. Wise, Stephanie Franz, Paul Kellam

المصدر: Wash, R, Calabressi, S, Franz, S, Griffiths, S J, Goulding, D, Tan, E-P, Wise, H, Digard, P, Haas, J, Efstathiou, S & Kellam, P 2012, ' Permissive and Restricted Virus Infection of Murine Embryonic Stem Cells ', Journal of General Virology, vol. 93, no. 10, pp. 2118-2130 . https://doi.org/10.1099/vir.0.043406-0Test
The Journal of General Virology

مصطلحات موضوعية: DNA Replication, Cell type, animal structures, Transcription, Genetic, viruses, Herpesvirus 1, Human, Biology, Virus Replication, Virus, Cell Line, Mice, Viral Proteins, 03 medical and health sciences, Dogs, 0302 clinical medicine, Orthomyxoviridae Infections, RNA interference, Cricetinae, Virology, Animals, Humans, RNA, Small Interfering, Embryonic Stem Cells, 030304 developmental biology, Mice, Knockout, 0303 health sciences, Innate immune system, Animal, Herpes Simplex, 11 Medical And Health Sciences, 06 Biological Sciences, Embryonic stem cell, Standard, 3. Good health, Mice, Inbred C57BL, Viral replication, Influenza A virus, Cell culture, Host-Pathogen Interactions, 07 Agricultural And Veterinary Sciences, Stem cell, DNA viruses, 030217 neurology & neurosurgery, HeLa Cells

الوصف: Recent RNA interference (RNAi) studies have identified many host proteins that modulate virus infection, but small interfering RNA ‘off-target’ effects and the use of transformed cell lines limit their conclusiveness. As murine embryonic stem (mES) cells can be genetically modified and resources exist where many and eventually all known mouse genes are insertionally inactivated, it was reasoned that mES cells would provide a useful alternative to RNAi screens. Beyond allowing investigation of host–pathogen interactions in vitro, mES cells have the potential to differentiate into other primary cell types, as well as being used to generate knockout mice for in vivo studies. However, mES cells are poorly characterized for virus infection. To investigate whether ES cells can be used to explore host–virus interactions, this study characterized the responses of mES cells following infection by herpes simplex virus type 1 (HSV-1) and influenza A virus. HSV-1 replicated lytically in mES cells, although mES cells were less permissive than most other cell types tested. Influenza virus was able to enter mES cells and express some viral proteins, but the replication cycle was incomplete and no infectious virus was produced. Knockdown of the host protein AHCYL1 in mES cells reduced HSV-1 replication, showing the potential for using mES cells to study host–virus interactions. Transcriptional profiling, however, indicated the lack of an efficient innate immune response in these cells. mES cells may thus be useful to identify host proteins that play a role in virus replication, but they are not suitable to determine factors that are involved in innate host defence.

وصف الملف: application/pdf

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::f984579056c8abd2bfa363f26b74276aTest
https://doi.org/10.1099/vir.0.043406-0Test

المؤلفون: Julia R. Gog, Edward C. Hutchinson, Paul Digard, Johann C. von Kirchbach

المصدر: Hutchinson, E C, von Kirchbach, J C, Gog, J R & Digard, P 2010, ' Genome packaging in influenza A virus ', Journal of General Virology, vol. 91, no. 2, pp. 313-328 . https://doi.org/10.1099/vir.0.017608-0Test

مصطلحات موضوعية: viruses, Orthomyxoviridae, Genome, Viral, Biology, ENCODE, medicine.disease_cause, Genome, Virus, 03 medical and health sciences, Orthomyxoviridae Infections, Virology, Influenza, Human, Influenza A virus, medicine, Animals, Humans, 030304 developmental biology, Genetics, 0303 health sciences, 030306 microbiology, Virus Assembly, Virion, RNA, biology.organism_classification, 3. Good health, Virion assembly, RNA, Viral, Function (biology)

الوصف: The negative-sense RNA genome of influenza A virus is composed of eight segments, which encode 12 proteins between them. At the final stage of viral assembly, these genomic virion (v)RNAs are incorporated into the virion as it buds from the apical plasma membrane of the cell. Genome segmentation confers evolutionary advantages on the virus, but also poses a problem during virion assembly as at least one copy of each of the eight segments is required to produce a fully infectious virus particle. Historically, arguments have been presented in favour of a specific packaging mechanism that ensures incorporation of a full genome complement, as well as for an alternative model in which segments are chosen at random but packaged in sufficient numbers to ensure that a reasonable proportion of virions are viable. The question has seen a resurgence of interest in recent years leading to a consensus that the vast majority of virions contain no more than eight segments and that a specific mechanism does indeed function to select one copy of each vRNA. This review summarizes work leading to this conclusion. In addition, we describe recent progress in identifying the specific packaging signals and discuss likely mechanisms by which these RNA elements might operate.

وصف الملف: application/pdf

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::e65318f0414bec0a2d7d47c19be14aaeTest
https://doi.org/10.1099/vir.0.017608-0Test

المؤلفون: Anne E. Mullin, Rosa M. Dalton, Paul Digard, Maria João Amorim, Debra Elton

المصدر: Journal of General Virology. 85:3689-3698

مصطلحات موضوعية: Viral Core Proteins, viruses, Orthomyxoviridae, RNA-Binding Proteins, RNA, Genome, Viral, Nucleocapsid Proteins, Biology, biology.organism_classification, Virology, Molecular biology, Virus, Cell Line, Nucleoprotein, Nucleoproteins, Ribonucleoproteins, Transcription (biology), Influenza virus nucleoprotein, Humans, RNA, Viral, Viral genome replication, Ribonucleoprotein

الوصف: Influenza virus genome replication requires the virus-encoded nucleoprotein (NP), partly because it is necessary to encapsidate the viral genomic RNA (vRNA) and antigenomic cRNA segments into ribonucleoproteins (RNPs). However, there is also evidence that NP actively regulates viral RNA synthesis and there is a long-standing hypothesis that increased concentrations of NP in the cell are responsible for a switch from genome transcription to replication. Here, this hypothesis is tested in a recombinant setting and in the context of virus infection. In a plasmid-based system for reconstituting active viral RNPs in cells, titration of increasing amounts of NP did not promote higher levels of genome replication relative to transcription, but in fact caused the opposite effect. An approximately fourfold reduction in the ratio of genomic and antigenomic RNAs to mRNA was seen across an 80-fold range of NP plasmid concentrations. When cells were transfected with the same amounts of NP plasmid to establish a concentration gradient of NP prior to virus superinfection, no change in the ratio of cRNA to mRNA was seen for segments 5 and 7, or for the ratio of segment 5 vRNA to mRNA. A slight reduction in the ratio of segment 7 vRNA to mRNA was seen. These findings do not support the simple hypothesis that increased intracellular concentrations of NP promote influenza virus genome replication.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::9d604c07f055a804989e9cc4726ac242Test
https://doi.org/10.1099/vir.0.80518-0Test

المؤلفون: Paul Digard, Agustín Portela

المصدر: Journal of General Virology. 83:723-734

مصطلحات موضوعية: viruses, RNA-dependent RNA polymerase, Karyopherins, Biology, Virus Replication, chemistry.chemical_compound, VP40, Transcription (biology), Virology, RNA polymerase, Viral Core Proteins, RNA-Binding Proteins, RNA, Biological Transport, Nucleocapsid Proteins, Orthomyxoviridae, Actins, Nucleoproteins, Ribonucleoproteins, chemistry, Viral replication, Helper virus, Influenza virus nucleoprotein, RNA, Viral

الوصف: All viruses with negative-sense RNA genomes encode a single-strand RNA-binding nucleoprotein (NP). The primary function of NP is to encapsidate the virus genome for the purposes of RNA transcription, replication and packaging. The purpose of this review is to illustrate using the influenza virus NP as a well-studied example that the molecule is much more than a structural RNA-binding protein, but also functions as a key adapter molecule between virus and host cell processes. It does so through the ability to interact with a wide variety of viral and cellular macromolecules, including RNA, itself, two subunits of the viral RNA-dependent RNA polymerase and the viral matrix protein. NP also interacts with cellular polypeptides, including actin, components of the nuclear import and export apparatus and a nuclear RNA helicase. The evidence for the existence of each of these activities and their possible roles in transcription, replication and intracellular trafficking of the virus genome is considered.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::af78260afa432a80a4762cbdf8215fa8Test
https://doi.org/10.1099/0022-1317-83-4-723Test