- Volume 1, Issue 1A, 2019
Volume 1, Issue 1A, 2019
- Oral Abstract
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- Virology Workshop: Clinical Virology
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High-throughput sequencing of patients with symptoms of unknown etiology
Routine clinical diagnosis of patients with a suspected viral infection involves screening with multiple assays, often limited to only a single genus or species. This approach, however, may fail to detect novel species, atypically presenting viruses (i.e a ‘respiratory’ virus causing neurological symptoms) and viruses that are imported from other countries; often due to clinicians focusing on the ‘likeliest’ candidates. High-throughput sequencing (HTS) allows for the identification viruses present within a sample, by sequencing all viral genomes present. Without the inherent bias of limiting screening targeted assays, these unusual viruses are more likely to be detected. Samples taken from patients with an illness of unknown etiology, were grouped into 5 pools; 2 Respiratory, 2 CSF and an EDTA blood pool. The CSF Pools each contained 200 samples, the Respiratory pools 100 and the EDTA blood pool 80. HTS libraries were created from each of these pools and an additional CSF sample from a single patient with encephalitis and were then sequenced using an Illumina HiSeq platform. Human Pegivirus was detected in both CSF Pools, the EDTA pool and a single respiratory pool. Picobirnavirus was detected in a respiratory pool. RT-PCR was used to screen individual samples compromising these pools. BK Polyomavirus and Mastadenovirus C were detected in the CSF of a patient who had presented with encephalitis. Coinfection of these viruses typically cause neurological symptoms only in immune-compromised patients, so this exemplifies the advantage of using HTS for the detection of atypically presenting viruses.
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Retrospective and prospective evaluation of chickenpox post-exposure prophylaxis (PEP) in at-risk groups before and after the change in PEP guidelines
More LessBackgroundPublic Health England (PHE) chickenpox post-exposure prophylaxis (PEP) guidelines were updated in August 2018. Varicella Zoster Immunoglobulin (VZIg) was replaced with oral aciclovir or valaciclovir in specific settings.
MethodWe retrospectively examined issues of VZIg between August 1 2017 and July 31 2018 at a transplantation centre that also had a large maternity service. In addition, since September 2018, information has been collected prospectively. Additional data were collected on exposure incident, patient risk group and any clinical or serological follow up.
ResultsThirty issues of VZIg were identified retrospectively over 12 months. These included 9 pregnant women and 21 immunosuppressed patients, average age was 7.6 years (median 4.4 years, SD 11.1). Prospective outcome data were collected since September 4 2018 involving 2 pregnant women and 6 immunosuppressed individuals. Clinical follow-up information was available for 2 pregnant women and 16 immunosuppressed patients. One pregnant woman exposed to her own child with chickenpox and 2 immunosuppressed patients developed clinical varicella at an average of 15.3 days post VZIg. Five immunosuppressed patients were VZV IgG negative post-exposure. Follow-up available to date for 2 of 8 exposure incidents after the change in guidance has shown no clinical or serological evidence of varicella infection.
DiscussionWhere follow up information was available, 19 % developed attenuated VZV around 2 weeks after receiving VZIg. Given the new guidance on using aciclovir prophylaxis, prospective outcome data are being collected and no VZV infections have been noted to date.
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- Virology Workshop: Gene Expression and Replication
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iPSCs derived from endothelial progenitors model latency and reactivation during human cytomegalovirus infection
To date, models of human cytomegalovirus (HCMV) latency and reactivation have depended on the use of primary myeloid cells, which have limited availability, are difficult to culture and are challenging to genetically modify. We now show that induced pluripotent stem cells (iPSCs) derived from circulating late outgrowth endothelial progenitors (EPC) can be differentiated down the myeloid lineage, where HCMV latent carriage and reactivation is known to occur in vivo, and act as a model to allow the interrogation of viral and cellular factors involved in latency and reactivation of this persistent human pathogen. In contrast, monocytes generated from iPSCs derived from de-differentiated fibroblasts failed to support HCMV latent carriage. These iPSCs derived from EPCs may also be suitable for in depth genetic interrogation of other viruses which also infect cells of the myeloid lineage, such as HIV and Zika.
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Identification of novel host factors influencing human cytomegalovirus replication using a two-step siRNA screen
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.
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Illuminating the molecular choreography of multi-segmented RNA genomes
More LessGenome segmentation offers certain evolutionary benefits to a number of pathogenic RNA viruses, including rotaviruses and influenza viruses. However, as the number of RNA segments per virion increases, the task of a non-random selection of a full set of distinct genomic RNAs poses a formidable challenge to maintaining the integrity of segmented genomes. Recently we have identified sequence-specific inter-segment interactions between rotavirus (+)ssRNA genome segment precursors. We have shown that binding of the rotavirus-encoded non-structural protein NSP2 to viral ssRNAs results in the remodeling of RNA, which is conducive to formation of inter-segment contacts. These protein-RNA interactions result in the stabilisation of extended intermolecular RNA-RNA contacts, potentially underpinning transient inter-segment interactions prior to genome encapsidation and replication. Using this approach, we have identified a number of RNA-RNA interaction sites in the rotavirus genome, which are likely to be involved in genome segment assortment process. Having established the role of NSP2 in promoting inter-segment RNA-RNA contacts, we have developed multiplexed imaging tools for directvisualization of the RNA assortment process in rotavirus-infected cells by employing single-molecule RNA FISH. To unravel the mechanisms, by which NSP2 controls the formation of inter-molecular RNA helices, we have applied RNA structure probing methods that allowed us to monitor conformational rearrangements, which are prerequisite for theformation of the RNA assortment complex. Our findings open up unique avenues for understanding the challenges for further improvement of the recently developed fully plasmid-based reverse genetics systems for rotaviruses.
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Infectious bronchitis virus modulates cellular stress granule signalling
More LessInfectious bronchitis virus (IBV), a gammacoronavirus, causes the economically important poultry disease, infectious bronchitis, resulting in reduced weight gain and egg quality. As observed for many viruses, during replication, IBV shuts off translation of host proteins, preventing synthesis of important products of the innate immunity, which are pivotal in fighting viral infection. This work investigates the role of stress granules in IBV translational control. Stress granules are membranes-less aggregations of stalled translation initiation complexes comprising translation initiation factors, 40S ribosome and RNA binding proteins. These structures serve as sites of storage and sequestration of translational machinery and cellular mRNA while simultaneously enabling intracellular signalling and antiviral responses. It is shown here by immunofluorescence that IBV induces stress granules in only a proportion of infected cells. These stress granules occur late in the virus life cycle and appear canonical, containing multiple stress granule markers and showing mRNA exchange with ribosomes. In addition, stress granule markers are not diverted to sites of virus replication, as seen during replication of some other viruses. Interestingly, IBV infection results in resistance to chemicals that induce stress granules via eukaryotic initiation factor 2α (eIF2α). Consistent with this, eIF2α is not phosphorylated at any time during IBV infection. This also indicates a non-canonical signalling pathway for IBV-induced stress granules. Significantly, stress granule formation is uncoupled from translational arrest as visualised using ribopuromycylation. Therefore, IBV replication both induces and inhibits cellular stress granule signalling in a process that is uncoupled from shut off of host translation.
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Replication of the Chikungunya virus genome requires cellular chloride channels
Chikungunya virus (CHIKV) causes fever and debilitating joint pain, with frequent long-term health implications and cumulating fatalities worldwide. There are no specific antivirals and vaccines, therefore understanding CHIKV replication is essential to establish treatments and preventative measures. Cellular ion channels are druggable targets and are known to facilitate replication of RNA viruses. To determine if the activities of cellular chloride channels (Cl--channels) are required during CHIKV replication, we applied broad-ranging inhibitors and siRNA to mammalian and invertebrate cells. The Cl--channel inhibitors DIDS, 9-ACA and NPPB significantly reduced the titre of released CHIKV progeny at 12 h post-infection in a dose-dependent manner suggesting that Cl--channels are pro-viral factors. Analysis of viral protein expression and time-of-inhibitor-addition studies indicated that CHIKV requires Cl--channels at post-entry and pre-egress stages. Replication of a sub-genomic replicon was restricted and genome copy numbers reduced by Cl--channel inhibition, implying that Cl--channels are involved in genome replication. siRNA knock-down identified the chloride intracellular channels (CLIC) 1 and 4 to be required for the CHIKV infectious cycle with CLIC1 interacting with the viral protein nsP3. We hypothesise that the channels play a role in formation or maintenance of the membranous, viral replication-complexes and that this important role is conservt amongst the mammalian and invertebrate hosts. These findings advance our understanding of CHIKV replication in the two host environments and help to identify drugs/druggable targets for treatment and prevention of CHIKV disease.
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Dimerisation of the influenza virus RNA polymerase during viral genome replication
Influenza virus encodes a heterotrimeric RNA-dependent RNA polymerase (RdRP), composed of subunits PB1, PB2 and PA, that carries out both transcription and replication of the viral RNA genome segments in the context of ribonucleoproteins. Replication of negative-sense viral RNA (vRNA) is a two-step process, progressing via a positive-sense complementary RNA (cRNA) intermediate. The mechanism of viral genome replication is mostly unknown, though there are multiple reports indicating RdRP dimerisation may be central for the process. Purified RdRPs from human and avian influenza A viruses both form dimers of heterotrimers in solution. Using a combination of X-ray crystallography, SAXS and cryo-EM, we identify the interface involved in RdRP dimerization, which is primarily located on the PA C-terminal domain. We use bimolecular fluorescence complementation (BiFC) to show that influenza RdRP forms dimers in mammalian cells through the interface identified in solution. Using a combination of cell-based and in vitro assays, we show that influenza RdRP dimerisation via the PA-C terminal domain is necessary for copying cRNA back into vRNA during viral genome replication. In addition, we show that a nanobody (a small-domain antibody) that interferes with dimerisation attenuates influenza A virus growth in cell culture. These data provide insight into the mechanism of influenza viral genome replication, and identify a potential novel drug target against influenza A virus.
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Encapsidation of viral RNA in Picornavirales: studies on cowpea mosaic virus demonstrate dependence on viral replication
More LessTo elucidate linkage between replication and encapsidation in Picornavirales, we have taken advantage of the bipartite nature of the plant-infecting member of the order, cowpea mosaic virus (CPMV), to decouple the two processes. RNA-free virus-like particles (eVLPs) can be generated by transiently co-expressing the RNA-2-encoded coat protein precursor (VP60) with the RNA-1-encoded 24K protease, in the absence of the replication machinery (Saunders et al., 2009). We have made use of the ability to produce assembled capsids of CPMV in the absence of replication to examine the putative linkage between RNA replication and packaging in the Picornavirales. We show here that the remarkable specificity of packaging observed in CPMV is due to a functional linking between the two processes of viral replication and encapsidation. We have created a series of mutant RNA-1 and RNA-2 molecules and have assessed the effect of the mutations on both the replication and packaging of the viral RNAs. We demonstrate that mutations that affect replication have a concomitant impact on encapsidation, and that RNA-1-mediated replication is required for encapsidation of both RNA-1 and RNA-2. This close coupling between replication and encapsidation provides a means for the specific packaging of viral RNAs. Moreover, we demonstrate that this feature of CPMV can be used to specifically encapsidate custom RNA by placing a sequence of choice between the RNA-2 sequences required for replication, which opens the door to novel research and therapeutic applications in the field of custom RNA packaging and delivery technologies.
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Identification of sites of Infectious Bronchitis Virus RNA synthesis
More LessInfectious bronchitis virus (IBV), an avian gammacoronavirus, is an important pathogen causing significant animal welfare problems and economic losses to the global poultry industry. Positive-strand RNA viruses, including coronaviruses, induce cellular membrane rearrangements during replication forming replication organelles, which are thought to support efficient viral RNA synthesis. IBV replication has been shown to induce the formation of double membrane vesicles (DMVs), zippered ER and tethered vesicles, known as spherules. Although these are proposed to be the site of viral RNA synthesis, this is as yet unconfirmed and is therefore the focus of these studies. Historically, dsRNA has been used as a marker for sites of coronavirus RNA synthesis, however IBV-associated dsRNA and nsp12 (the viral RNA-dependent RNA polymerase) do not colocalise in infected cells. We have determined the cellular location of the viral genome using Fluorescence In Situ Hybridisation (FISH). By comparing the immunofluorescence labelling of cells permeabilised with different detergents, we have demonstrated that dsRNA can be found within membrane-protected compartments, while nsp12 is not, indicating that the virus could be isolating the dsRNA in DMVs or spherules, affording protection from the host immune response. By incorporating uridine analogues over the course of infection with IBV, we have visualised sites of nascent viral RNA synthesis using super-resolution microscopy. This has shown that dsRNA appears to colocalise more strongly with nascent RNA than nsp12. Using these methods as well as looking at the ultrastructural level we are able to begin to discover the location of sites of IBV RNA synthesis.
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The role of RNA-RNA interactions in the assembly and reassortment of influenza A viruses
Influenza A virus has a genome consisting of 8 segments of negative sense RNA. When two influenza A virus strains infect the same cell, there is potential for the progeny to package segments from both strains. This process is termed reassortment and can lead to rapid genetic shifts that have previously generated strains of influenza responsible for pandemic events. Recent evidence suggests that assembly of the eight influenza genomic segments for packaging into a virion is mediated by RNA-RNA interactions between the segments. These interactions are likely to contribute to the varying compatibilities for reassortment observed between segments from different strains of influenza. We have captured complete RNA-RNA interaction maps for several influenza A viruses using a high-throughput sequencing approach and identify extensive, redundant, networks of RNA-RNA interactions between the genomic viral RNA segments. We extended this analysis to H1N1 and H3N2 reassortants, and found that by manipulating these interactions, we can drive preferential co-segregation of segments during reassortment. This work provides the first direct evidence that RNA-RNA interactions between the influenza virus genomic segments are a key factor in driving reassortment between viral strains.
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Investigating the impact of Herpes simplex virus type 1 latency-associated non-coding RNAs on apoptosis in human neuronal cells
More LessHerpes simplex virus 1 (HSV-1) establishes latency in sensory neurons, allowing it to persist for the lifetime of the host. During latency, the only abundantly transcribed HSV-1 gene is the latency-associated transcript (LAT), which is processed into the 2.0 kb major LAT intron and several microRNAs. These non-coding RNAs (ncRNAs) have been reported to influence latency, possibly through limiting apoptosis of infected cells. As these studies have used animal models or non-neuronal cell culture, we have developed a differentiated human neuroblastoma (SH-SY5Y cells) model to examine their effect in human neuronal cells. We have infected these neuronal cultures with replication-defective HSV-1, which establishes a quiescent infection and strongly expresses the latency ncRNAs. We show that quiescent HSV-1 infection reproducibly protects differentiated SH-SY5Y from etoposide-induced apoptosis. We are also further defining the contribution of different LAT ncRNAs using recombinant lentiviruses to drive expression of the LAT intron or microRNAs. Furthermore, we are also currently exploring the mechanisms of this anti-apoptosis effect, and broader virus-neuron interactions by characterising whether the human neuronal transcriptome is altered by LAT RNA expression. Improving our understanding of the molecular interactions underpinning HSV-1 latency in neurons could help develop novel therapies to target HSV-1 latency.
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Human papillomavirus E6 regulates the trafficking of gap junction protein Cx43
More LessHigh-risk human papillomavirus (HR-HPV) infects epithelial cells and is the major cause of anogenital and oropharyngeal cancers. HR-HPV oncogenic activity is through E6 control of p53, but E6 binds and degrades PDZ proteins such as the tumour suppress protein hDlg (human homologue of DrosophilaDiscs Large). The E6/hDlg complex also contains Connexin 43 (Cx43) (MacDonald, Sun et al. 2012), the major building block of gap junctions that allow intercellular molecular communication. In HPV16-positive non-tumour cervical keratinocytes (W12NT: low E6 levels) Cx43/hDlg is on the plasma membrane but in the cytoplasm in W12T tumour epithelial cells (W12T: high E6 levels) correlating with loss of gap junction cell-cell communication. E6 siRNA depletion in W12T cells restored Cx43 to the cell membrane, while overexpressing E6 in HPV-negative cervical cancer cells C33a resulted in Cx43 moving to the cytoplasm. E6 could control Cx43 trafficking through controlling hDlg or by altering cell signalling. In the absence of E6 in HEK293, HaCaT and normal immortalised keratinocytes (NIKS), Cx43 and hDlg could be co-immunoprecipitated and they co-localised on the plasma membrane. Thus, the Cx43/hDlg interaction is not carcinoma cell-specific, is not dependent on HR-HPV E6, and may have a functional role in non-cancer cells. siRNA depletion of hDlg, led to reduction in Cx43 protein levels and some relocation to the cytoplasm. This indicates that HPV E6 controls Cx43 through interaction with and degradation of hDlg. However, we cannot discount that E6 itself may have additional effects on Cx43 levels and trafficking besides via hDlg.
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Single cell and single molecule resolution of herpesvirus genome transport, condensation state and transcriptional output
More LessEvents controlling herpesvirus nuclear genome uncoating, nuclear transport, and the onset of transcription remain poorly understood. We have now developed procedures to examine these processes within individual cells and at the single molecule level for both the genome and the transcripts produced from it. We have combined two novel techniques of, firstly, bioorthogonal chemistry to visualise genomes which incorporate an alkyne-nucleoside analogue (ethynyl deoxycytidine, EdC) and secondly, single molecule RNA in-situ hybridisation (smFISH) which allows detection of individual mRNA transcripts. Using these techniques simultaneously, we can now qualitatively and quantitatively analyse individual transcript abundances and their intracellular localisation, in relation to the genome itself at single molecule resolution during the progression of infection. Moreover, we are able to examine these parameters when a single genome infects a cell. We have examined the transcripts of the immediate-early mRNA for ICP0, and features revealed from this work include; transcriptional ‘bursting’ with clustered transcripts around individual genomes; mean mRNA transcript number, variance, and intracellular localisation produced from a single genome; the progressive abundant ICP0 transcription occurring selectively from replicated genomes; an increasing bottleneck in cytoplasmic transport of transcripts emanating from replicated genomes; and increased transcription bursts from virtually every uncoated genome when protein synthesis is suppressed. Further, by multiplexing probes, we can simultaneously analyse distinct transcription outputs from different genes of the same or classes, and genomes, in the same individual cell. Our results reveal completely new perspectives on the very early events of genome presentation and transcription from those genomes.
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Developing a universal strategy for cloning and assembly of the genomes of diverse Epstein – Barr virus strains
More LessEpstein-Barr virus (EBV), is an oncogenic gamma-herpesvirus, which is associated with malignant diseases of B cells, T cells, and epithelial cells. EB viruses have large DNA genomes of more than 170 kb that are difficult to clone and manipulate. Here we describe 2 different approaches for cloning whole EBV genomes of diverse strains for reverse genetics studies. The first approach used CRISPR/Cas9-mediated cloning of the entire EBV genome into a bacterial artificial chromosome (BAC) vector using homologous recombination in B cells. This method allowed the cloning of the type 2 EBV strain Jijoye for the first time, but the BAC-clones are unstable. This strategy is being modified by recoding the homology regions to make the clones more stable. The second approach involves transformation-associated recombination (TAR) cloning of EBV fragments and their assembly in yeast, which will allow for mixing and matching DNA regions from different EBV strains for functional studies. This approach is based on TAR cloning of the EBV genome as 10 overlapping fragments, which average 17 kilobases long, using the natural homologous recombination processes of the yeast. Subsequent assembly of all the overlapping fragments is undertaken in yeast or by Gibson assembly to reconstitute the infectious EBV clone. Two fragments from EBV strains B95-8 and AG876 were captured and isolated successfully, but at low efficiency. We are currently improving the TAR cloning efficiency by increasing the size of the capture homology regions to approximately 500 bp coupled with CRISPR/Cas-9-mediated fragmentation of the EBV genome.
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The effects of APOBEC3 proteins on Hepatitis B virus replication
More LessHepatitis B virus causes chronic liver infection in 257 million people worldwide. Current treatments against HBV can control, but not cure HBV infection. Therefore, new treatments for chronic HBV infection need to be developed. The APOBEC3 (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3) proteins are cellular restriction factors, which have been shown to restrict viral replication for a number of viruses (e.g. HIV) and for retrotransposons. The aim of this study is to elucidate the role of APOBEC3 proteins in inhibiting Hepatitis B virus replication. Quantitative polymerase chain reaction (qPCR) was used to evaluate the impact of APOBEC3 family members on the Hepatitis B virus replication in HepG2.2.15 cells. The highest inhibition of intracellular capsid associated HBV DNA, extracellular virion associated HBV DNA was induced by APOBEC3DE, APOBEC3F, and APOBEC3G as compared to the other APOBEC3 proteins. However, APOBEC3DE showed no inhibition of HBV total RNA, whereas the highest inhibition of HBV total RNA was induced by APOBEC3F and APOBEC3G. The sub-cellular localisation of APOBEC3 proteins was determined by immunofluorescence using confocal microscopy. It was found that APOBEC3DE, APOBEC3F, and APOBEC3G localise to the cytoplasm, suggesting a crucial role of these proteins in HBV replication in the cytoplasm. Nevertheless, APOBEC3A and APOBEC3B localise to the nucleus. The expression and co-localisation of APOBEC3 proteins and viral and host proteins (Uracil DNA Glycosylase UNG and ATP-dependent RNA helicase DDX3) in HepG2.2.15 and HEK293T cells are being investigated in order to determine the interaction between these proteins.
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- Virology Workshop: Innate Immunity
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Structural elucidation of viral antagonism of innate immunity: the STAT1 interface
To evade innate immunity, many viruses express interferon-antagonists that target STATs, critical mediators of immune signalling. Virus-STAT interfaces may provide new therapeutic targets but progress is hindered by a lack of direct structural data, owing to poor tractability of antagonists/full-length STATs for structural/biophysical approaches. By applying cross-saturation transfer NMR, we report the first direct structural analysis of binding of full-length STAT1 to an interferon-antagonist of a human pathogenic virus, the first such study of the virus-host interface. Analysis using mutation of the interface, biophysical characterization, immune signalling/protein-protein interaction assays including PCA, reverse genetics and animal infection demonstrated the significance of this interface in immune signaling suppression, and in disease caused by a pathogenic field-strain lyssavirus. Importantly, NMR/mutagenesis also revealed that the interface comprises multiple surfaces/domains in both the viral and cellular partners, indicating that antagonism involves extensive interactions consistent with a multifaceted inhibitory mechanism, distinct from ‘simple’ mechanisms such as tethering. Furthermore, by elucidating the spatial relationship of interactions critical to immune evasion and replication, the data provide insight into how ostensibly simple viruses can regulate these central functions via a single multifunctional protein. These data provide novel insights into fundamental viral biology, and potential exploitation of these mechanisms as new targets for antivirals and vaccine development. The study also demonstrates the power of biophysical/NMR approaches to elucidate the atomic interface of full-length STATs with regulatory proteins, providing a framework for studies to reveal immune evasion mechanisms of other pathogens in their full complexity.
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HIV-1 Vpr accessory protein interacts with REAF and mitigates its associated anti-viral activity
The accessory protein Vpr of Human Immunodeficiency Virus type 1 (HIV-1) enhances replication of the virus in macrophages. Virus particle packaged Vpr is released in target cells shortly after entry, suggesting it is required early in infection. Why it is required for infection of macrophages and not cycling T-cells and why it induces G2/M arrest in cycling cells are unknown. Here we observe, by co-immunoprecipitation assay, an interaction between Vpr and endogenous REAF (RNA-associated Early-stage Antiviral Factor, RPRD2), a protein shown previously to potently restrict HIV infection. After HIV-1 infects macrophages, within 30 min of viral entry, Vpr induces the degradation of REAF. Subsequently, as replication continues, REAF expression is upregulated – a response which is curtailed by Vpr. REAF is more highly expressed in differentiated macrophages than in cycling T-cells. Expression in cycling cells is cell-cycle dependent and knockdown induces cell-cycle perturbation. Therefore, our results support the long held hypothesis that Vpr induces the degradation of a factor involved in the cell cycle that impedes HIV infection in macrophages.
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Human Cytomegalovirus pUL83 targets core histones to inhibit interferon synthesis and promote viral spread
More LessTegument protein pUL83 is the most abundant component of human cytomegalovirus (hCMV) particles. The viral protein is predicted to be composed of three domains: a pyrin association domain (PAD), a carboxy-terminal domain (CTD), and an intrinsically disordered linker domain (amino acids 388–479) located between the PAD and CTD. Although pUL83 has been shown to antagonize interferon (IFN) responses, it has not been fully elucidated how the viral protein may contribute to hCMV replication. In this study we demonstrate that pUL83 associates broadly with viral and host chromatin including condensed chromosomes during mitosis. We further show that the linker domain in pUL83 is both required and sufficient for host chromatin targeting, and that this interaction depends on two evolutionary conserved arginine residues (R453 and R455) in the viral protein. Our data indicate that the pUL83 linker domain specifically associates with human core histones (but not linker histones). Furthermore, pUL83 inhibits IFN-beta and IFN-lambda gene induction, but not expression of other cytokine genes, via a mechanism that largely depends on the linker domain including R453/455. Although earlier studies suggested that pUL83 is dispensable for productive hCMV infection in fibroblasts, we find that the viral protein is necessary for efficient plaque formation in these cells, specifically in the presence of IFN. Finally, the pUL83 linker domain including R453/455 contributes significantly to the plaque size in hCMV-infected fibroblasts. Overall, we propose that pUL83 promotes spread of hCMV by selectively inhibiting induction of IFN gene expression via a novel chromatin-based molecular mechanism involving core histones.
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Type I interferon activity promotes a cellular environment that supports the establishment of latency by human cytomegalovirus
Type I interferons (IFN) are potent inducers of an anti-viral state in response to infection and have been demonstrated to inhibit cytomegalovirus (CMV) replication both in vitro and in vivo. CMV, like all herpes viruses, has the capacity to establish lifelong infections of host through the establishment of latency. As the very early stages of viral entry can trigger IFN responses we investigated the impact of IFN on the establishment of latent human CMV (HCMV) in myeloid progenitor cells. Here we show that priming of myeloid THP1 cells with type I IFN prior to infection skews infection towards a more efficient establishment of latency. This is evidenced by detection of reduced lytic gene expression, increased latent gene expression, and increased levels of reactivation following differentiation. Blockade of IFN signalling with neutralising antibodies antagonised the latent phenotype suggesting that endogenous IFN production upon infection contributed to the effect observed. Intriguingly, whilst both IFNα2 and IFNβ can drive latent infection individually, their effects were dose-dependent and demonstrated a biphasic impact on the establishment of latency, with the highest doses of IFN preventing both lytic and latent infection. These data demonstrate that the HCMV derives an unexpected benefit from IFN production. They support a hypothesis that, although anti-viral in nature, concentration-specific effects of IFN may be evident in the cells which can modulate different outcomes post infection in persistent viruses such as HCMV. Future work is identifying the IFN concentration-specific effects responsible for a cellular environment that favours the establishment of latency.
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Characterization the role of key DNA sensors in herpes simplex virus replication
More LessDNA sensors including cGAS, STING and IFI16 are key components of the innate immune response to infection. However, the precise mechanisms of action, in particular the relative importance of direct suppression of replication versus paracrine signalling of an antiviral state to susceptible cells remains unclear. We examined the kinetics of herpes simplex virus infection and spread in a relevant cell type, human keratinocytes, lacking one or other of these DNA sensors using time-lapse microscopy. We also examine transcriptional induction of interferon from the native locus, at single cell and single molecule level using highly sensitive RNA FISH. Our results reveal distinct aspects of the roles of these factors and reveal outcomes not appreciated by other methods. Cells lacking either of these factors showed increased susceptibility to initial infection (prior to any downstream paracrine signalling) but with quite different outcomes. Lack of cGAS resulted in increased cellular migration and cell density at the infection focus. On the other hand, cells lacking STING showed lower cell density and significantly increased cytopathic effect likely curtailing virus yield. Initial results demonstrate that we can analyse interferon transcription at single cell level with exquisite sensitivity down to a few transcripts per cell and reveal profound spatial heterogeneity in responses to induction by PAMP ligands. Altogether, our results reveal new insight into the spatial landscape of the initiation and spread of HSV and key cellular responses which likely integrate pathways including innate immunity, apoptosis and cell migration.
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The African horse sickness virus NS4 counteracts the antiviral response and is a determinant of viral virulence
More LessAfrican horse sickness is a major infectious disease of equids and is caused by African horse sickness virus (AHSV), a dsRNA virus with 10 genome segments encoding for 7 structural and 4/5 non-structural proteins. Here, we focused on the characterisation of the AHSV NS4, the latest protein found to be expressed by this virus. In silico analysis of available sequences confirmed the existence of two phylogenetically distinct AHSV clades: NS4-I and NS4-II. NS4-II is further divided into three subtypes (a, b and g). Confocal microscopy demonstrated that all AHSV NS4 types localised in the cytoplasm of infected cells, unlike the BTV NS4 which, has a strong nucleolar localisation. The replication kinetics of reverse genetics derived AHSV NS4 deletion mutants (AHSVDNS4) were similar to their wild type counterparts in insect (Kc) or interferon incompetent (BSR) cells. However, replication of AHSVDNS4 mutants in primary horse endothelial cells was restricted, in comparison to wild-type viruses. Importantly, primary cells restriction to AHSV replication was dependent on the JAK/STAT pathway. Furthermore, AHSVDNS4 mutants were not able to efficiently suppress the secretion of anti-viral cytokines from primary cells, while the wild-type viruses suppressed this response to varying degrees. Importantly, AHSVDNS4 mutants were less virulent than their wild type counterparts in a murine model of AHSV infection. These results indicate that AHSV NS4 has a role in interferon IFN antagonism and a determinant of viral virulence. We are currently carrying out mass spectrometry analyses to identify the cellular proteins interacting with the AHSV NS4.
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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
RationaleThe 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.
ObjectiveTo investigate the impact of hRSV on the secretome of pediatric respiratory epithelium.
MethodsA 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 resultsFollowing 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.
ConclusionshRSV 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.
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The long isoform of ZAP widely restricts Paramyxoviruses
More LessParamyxoviruses (PVs) are negative-sense RNA viruses that are important in human and animal health, and can cause severe zoonotic diseases. We screened a wide range of animal and human PV matrix proteins for host interactions, and found that the long (L) isoform of the host protein ZAP (zinc-finger antiviral protein) interacted with all the tested viral matrixes. ZAP-L is constitutively expressed, has a prenlyation motif for membrane-localization, and primarily mediates antiviral activity by binding and degrading viral RNAs through the exosome complex. However, ZAP restriction of PVs has not been demonstrated in the literature. We found that knockdown of ZAP-L results in increased replication of a panel of five human and animal PVs. Overexpression of ZAP-L (but not ZAP-short) restricts replication of PVs in a reciprocal pattern – with the notable exception of Sendai virus (SeV) – and as with other viruses, mutating the prenylation motif of ZAP-L abolishes restriction. RT-qPCR of PV RNAs and pulled-down RNAs does not indicate specific targeting of a viral transcript by ZAP-L, although overall genome abundance is reduced. Finally, immunoprecipitation of ZAP shows an additional RNA-independent interaction between ZAP-L and SeV-nucleocapsid not found with HPIV3-nucleocapsid, a closely-related PV. Thus, we have observed that ZAP-L interacts with the matrix of, and restricts replication of, a wide range of paramyxoviruses. A PV that is not restricted (SeV), has an additional interaction with ZAP via its nucleocapsid protein that may ameliorate ZAP restriction. Investigating ZAP-related restriction differences between closely-related PVs may shed light on anti-viral mechanisms of ZAP.
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Investigating the interferon antagonistic abilities of louping ill virus, a neglected animal pathogen endemic to the UK
More LessLouping ill virus (LIV; Flavivirus, Flaviviridae) is an important—but poorly characterized—animal pathogen of significant economic concern within the UK. Transmitted by ticks, LIV predominantly causes disease in ruminants and grouse, resulting in heavy losses. LIV is closely related to another flavivirus—tick-borne encephalitis virus (TBEV) which, unlike LIV, is a significant human pathogen. The molecular mechanisms that underpin host restriction in these viruses are poorly understood however, previously it has been shown that the TBEV non-structural (NS) proteins do not act as type-I interferon (IFN) antagonists, unlike many other Flavivirus NS proteins. Therefore, to facilitate comparison with LIV we investigated the possible antagonistic actions of the LIV NS proteins using a luciferase-based IFN reporter assay. Utilising this assay we identified six LIV NS proteins that function as antagonists throughout the IFN induction cascade. We also identified and modelled a subgenomic flavivirus RNA (sfRNA) that is produced during LIV infection and is similar in structure to TBEV sfRNA. We found that the LIV and TBEV sfRNAs antagonise RIG-I, indicating that the IFN antagonistic ability of sfRNA is not limited to mosquito-borne flaviviruses. Finally, we established the first LIV reverse genetics system using circular polymerase extension reaction (CPER). This powerful tool can be used to produce chimeric viruses which will allow further investigation into the factors governing host restriction and virulence in tick-borne flaviviruses. Investigating the mechanisms that underlie LIV infection aids our understanding of interferon antagonism in flaviviruses and the molecular determinants of host restriction.
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- Virology Workshop: Morphogenesis, Egress and Entry
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Optimisation of an in vitro cell system using pseudoviruses to investigate HBV entry mechanisms
More LessAimTo explore the roles of large (L), middle (M) and small (S) surface antigens in an in vitro model of hepatitis B virus (HBV) entry, and thereby to achieve an optimal in vitro cell system using pseudoviruses to investigate HBV entry mechanisms.
MethodsRNA encoding sodium taurocholate co-transporting polypeptide (NTCP) was extracted from human hepatocytes and cloned into the pHIV-EGFP expression vector. The resulting pHIV-NTCP-EGFP construct was delivered into Huh7 hepatoma cells with the aid of pCMVR87.4 (packaging vector) and pCMV.VSV.G (glycoprotein), subsequently, a cell line over-expressing NTCP was generated. Meanwhile, by silencing start codons at L, M or S, seven constructs were obtained, i.e. L + M -S-, L-M + S -, l -M-S+, L + M + S-, L-M + S +, L + M -S+, l -M-S-. A matrix with various amounts of the seven constructs was used to generate HBV pseudoparticles (HBVpp) using a Luciferase-based HIV (pNL4.3.luc.R-E-) pseudotype entry model system. The infectivity of the HBVpp was tested in NTCP naïve and NTCP over-expressing Huh7 hepatoma cells.
ResultsThe relative amounts of L, M and S were critical in determining the efficiency of entry of HBVpp into NTCP + ve Huh7 cells.
ConclusionsCreation of NTCP-over expressing cells together with optimisation of conditions to maximise HBVpp entry provides an important tool to investigate the entry step in the HBV life cycle, and may allow identification of non-NTCP-dependent viral entry pathways.
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Characterising Birnaviridae replication and reassortment in vitro: virus factories derived from distinct input viruses form in the cytoplasm of co-infected cells and coalesce over time
More LessThe Birnaviridae family is comprised of non-enveloped viruses with a double-stranded RNA genome that is divided into two segments, A and B. Birnaviruses are responsible for major economic losses to the poultry and aquaculture industries, and reassortment complicates their epidemiology and control. However, little is known about the nature of theirreplication in cells, or the molecular mechanism underpinning reassortment. In order to address this, we rescued two recombinant infectious bursal disease (IBD) viruses, with either a GFP11 or Tetracysteine (TC) tag at the 3’ end of segment B (IBDV-GFP11 and IBDV-TC, respectively). DF-1 cells were either transfected with GFP1-10 prior to IBDV-GFP11 infection, or stained with ReAsH following IBDV-TC infection, which led to the apprearance of green or red foci in the cytoplasm, respectively. Foci co-localised with VP3 and dsRNA, suggesting these were virus factories (VFs). The average number of VFs significantly decreased from 60 to 5 per cell between 10 and 24 h post infection (P<0.01), while the average area significantly increased from 1.24 µm2 to 45.01 µm2 (P<0.01), suggesting VFs coalesce in the cytoplasm over time. Red, green and yellow foci were observed in the cytoplasm of co-infected cells, suggesting that VFs are initially derived from distinct input viruses prior to coalescence. Live cell imaging revealed that larger VFs were more static while smaller VFs were more mobile, and fusion events were observed. We speculate that VF coalescence is required for birnavirus reassortment, and current work is aimed at determining the cellular factors that drive coalescence.
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A high throughput siRNA screen to identify host membrane trafficking proteins that restrict pneumovirus egress
More LessThe pneumoviruses human and bovine respiratory syncytial virus (RSV) are significant global respiratory viral pathogens responsible for causing lower respiratory tract infections in humans and cattle, respectively. The formation of progeny virions at the cell surface requires the coordinated assembly of glycoproteins trafficked to the apical surface of cells via the host secretary pathway, other viral protein complexes assembled in cytoplasm inclusion bodies and lastly viral ribonucleoproteins. Although some host proteins and pathways have been implicated in pneumovirus budding and assembly (e.g. HSP90, Rab-11 and apical recycling endosomes), the full spectrum of virus-host interactions has not been fully elucidated. Using a siRNA library targeting membrane trafficking and the Incucyte® Live Cell Imaging System, we have developed and optimised a high-throughput siRNA protocol for characterising RSV egress. The siRNA library targets human proteins which are known, or predicted, to be involved in membrane trafficking or remodelling, while the Incucyte® System allows near real-time imaging over an extended time-course, generating high quality data that allows the monitoring and quantification of multiple parameters such as cell viability, viral replication and syncytia formation. Using a recombinant human RSV expressing a GFP reporter we have identified a number of proteins involved in pneumovirus trafficking in infected cells. Our techniques provide a robust and sensitive mechanism for genetic screening and the identification of pneumovirus-protein interactions.
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Mapping the pH sensors critical for host cell entry by a complex Nonenveloped Virus
Weining Wu and Polly RoyBluetongue virus (BTV), in family Reoviridae, is an insect-borne, double capsid virus causing haemorrhagic disease in livestock around the world. Here, we elucidate how outer capsid proteins VP2 and VP5 coordinate cell entry of BTV. The recently solved high-resolution structures reveal unique features of BTV VP2 and VP5. To identify key functional residues, we used atomic-level structural data to guide mutagenesis of VP2 and VP5 and a series of biological and biochemical approaches, including site-directed mutagenesis, reverse genetics-based virus recovery, expression and characterization of individual recombinant mutant proteins, and various in vitro and in vivo assays. We demonstrate the dynamic nature of the conformational change process, revealing that a unique zinc finger (CCCH) in VP2 acts as the major low pH sensor, coordinating VP2 detachment, subsequently allowing VP5 to sense low pH via specific histidine residues at key positions. We show that single substitution of only certain histidine residues has a lethal effect, indicating that the location of histidine in VP5 is critical to inducing changes in VP5 conformation that facilitates membrane penetration. Further, we show that the VP5 anchoring domain alone recapitulates sensing of low pH. Our data reveal a novel, multiconformational process that overcomes entry barriers faced by this multicapsid nonenveloped virus.
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Investigating the mechanisms of BK polyomavirus egress and virus-host interactions
More LessBK polyomavirus (BKPyV) is a small, non-enveloped dsDNA virus that can establish a lifelong, silently persistent infection in the kidney and is estimated to infect 70–90 % of the world’s population. In immunocompromised individuals, particularly bone marrow and kidney transplant patients, increases in BKPyV replication can result in significant pathological conditions. In the case of kidney transplant patients, this can result in nephropathy, which in severe cases can result in the deterioration of allograft function and loss of the transplanted organ. There are currently no antiviral treatments with strong evidence of clinical efficacy against BKPyV. Though little is known about BKPyV egress from infected cells, we have evidence showing that BKPyV can be released in a non-lytic manner by an unconventional cellular secretory pathway that bypasses the Golgi apparatus. Here, we investigate the mechanisms behind BKPyV non-lytic egress through studying the effects of knocking out candidate host proteins involved in unconventional secretory pathways on BKPyV release, examining the effects of BKPyV infection on host cell protein secretion, and ascertaining which host proteins are essential for the BKPyV life cycle via a whole-genome CRISPR screen. These experiments are uncovering novel virus-host interactions that, when targeted, could lead to antiviral effects.
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Characterisation of new environmental bacteriophages targeting the Escherichia coli LamB outer membrane porin
More LessBacteriophages are viral parasites of bacteria. A successful infection starts with the adsorption of the bacteriophage to a specific receptor on the host cell surface. Most bacteriophages are thought to have a narrow host range but this can be extended in certain cases. One strategy forextending host range is to first express a known functional bacteriophage receptor protein in bacteria previously non-susceptible to the bacteriophage, thereby enabling adsorption and potential infection by viruses that target the specific receptor. To investigate the feasibility of this approach, a plasmid (pMUT13) encoding the Escherichia coli LamB porin, the receptor for bacteriophage Lambda, was transferred into three different enterobacterial genera, namely Citrobacter, Yersinia, and Serratia. Over 100 environmental bacteriophages were isolated that infected these pMUT13-containing strains, and some bacteriophages were shown to infect their respective hosts in a LamB-dependent way. The host ranges of the environmental bacteriophages were cross-tested across the heterologous genera and surface adsorption kinetics investigated. Unlike bacteriophage Lambda, which is a member of the Siphoviridae, these newly-isolated LamB-dependent bacteriophages were more commonly members of the Myoviridae, based on transmission electron microscopy and whole genome sequences. Furthermore, an interesting selection of evolved bacteriophage mutants with broader host range were isolated, and the key mutations involved in their evolution to adapt to new hosts were investigated by genome analysis.
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Generation, lyophilisation and epitope modification of high titre filovirus pseudotyped lentiviruses for use in antibody neutralisation assays and ELISA
The 2014–2016 Ebola outbreak in West Africa highlighted the need for improved diagnostics, surveillance and therapeutics for filoviruses. The need for high containment virus handling facilities creates a bottleneck hindering research efforts. A safe alternative to working with native viruses are pseudotyped viruses (PV) which are non-replicating particles bearing surface glycoprotein(s) that can be used for antibody detection. The aim of this study was to create a diagnostic tool to distinguish between genera and species of pathogenic filoviruses (e.g. neutralization tests and ELISA), avoiding the cross reactivity currently seen. High titre PVs bearing the receptor glycoprotein (GP) of different filovirus species, plus specific epitope chimeras, were successfully generated. Next, lyophilisation studies to assess particle stability/degradation transportation and long-term storage were conducted. Filoviruses maintained their titres for at least 1.5 years after lyophilisation when kept in temperatures of up to 4 °C, with all filovirus genera following a similar trend. At higher temperatures, PVs degraded to unworkable titres. Reconstituted PVs also performed well in neutralisation assays. A chimeric cuevavirus GP bearing ebolavirus (Zaire sp.) epitopes KZ52 and 1 H3 retained infectivity, with average titres of approximately 1×10 7 RLU ml−1, similar to wild type, indicating its structure was not compromised. These chimeras are now being assessed in neutralisation tests using specific monoclonal antibodies and incorporated into ELISA with PVs as antigens. The data suggests lyophilised PVs are amenable to long-term storage, and their GPs can be modified to create artificial antigens for diagnostics and serosurveillance.
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Using cryo-electron tomography to elucidate the structural basis of the endosomal K+ requirement during Bunyavirus entry
More LessThe Bunyavirales order are the largest group of negative stranded RNA viruses, infecting humans as well as a bewildering array of animals and plants, in which select members cause severe or fatal disease. To enter host cells, bunyaviruses undergo endosomal transport to specific cellular destinations and exploit the changing environment of maturing endocytic vesicles to mediate genome release. Several virus-endosome fusion triggers have previously been identified, including endosomal potassium (K+) recently identified by our group. Specifically, we demonstrated a role for K+ channels and endosomal K+ concentration ([K+]) in the ‘priming’ of virions for fusion and uncoating events. Interestingly for Bunyamwera virus (BUNV), both a reduced pH and elevated [K+] were required to permit endosomal escape of the virus. To understand the molecular basis for this requirement we have used cryo-electron tomography to study the changes in virion structure upon K+ and pH treatment. These studies reveal why endosomal [K+] and K+ channels are required for bunyavirus entry, highlighting the potential of K+channels as druggable anti-viral targets.
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Cellular cholesterol abundance regulates potassium accumulation within endosomes and is an important determinant in Bunyavirus entry
The Bunyavirales order of segmented negative sense RNA viruses includes over 500 isolates that infect insects, animals, and plants, and are often associated with severe and fatal disease in humans. To multiply and cause disease, bunyaviruses must transport their genomes from outside the cell into the cytosol, achieved by transit through the endocytic network. We have previously shown that the model bunyaviruses Bunyamwera virus (BUNV) and Hazara virus (HAZV) exploit the changing potassium concentration ([K+]) of maturing endosomes to release their genomes at the appropriate endosomal location. K+ was identified as a biochemical cue to activate the viral fusion machinery, promoting fusion between viral and cellular membranes, consequently permitting genome release. In this study, we further define the biochemical prerequisites for BUNV and HAZV entry and their K+ dependence. We report four major findings: (1) BUNV and HAZV require cellular cholesterol during virus infection; (2) cholesterol is required during BUNV endosomal escape; (3) cholesterol depletion from host cells impairs their ability to accumulate K+ in maturing endosomes, revealing new insights into endosomal K+ homeostasis; (4) ‘priming’ BUNV virions with K+ prior to infection alleviates BUNV cholesterol requirement, revealing the mechanism of cholesterol dependence. Taken together, we provide a new model in which cholesterol abundance influences K+ endosomal homeostasis and consequently the efficiency of bunyavirus infection. The ability to inhibit bunyaviruses with existing cholesterol lowering drugs offers new options for future anti-bunyavirus interventions for pathogenic family members.
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- Virology Workshop: Pathogenesis
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Inter-species transmission of avian influenza virus to dogs: 10 years experience
More LessInfluenza viruses have continuously evolved into multiple mutant strains from several regions, resulting in aggravated endemic or epidemic outbreak conditions. In the 2000s, several outbreaks of inter-species transmission were reported, such as, the avian H3N2 influenza virus that crossed the host barrier to dogs. The inter-species transmission gave rise to the H3N2 canine influenza virus (CIV) that spread from East Asia to North America. The newly emerged H3N2 CIV was likely to infect to cats; however, ferrets, which had a SA receptor-binding pattern similar to that of humans, were not suitable natural hosts. In addition to avian-to-dog transmission, the infectivity of pdm H1N1 and seasonal H3N2 viruses in dogs was proven when artificial inoculation of the viruses with active viral shedding in dogs caused pathologic changes in the lungs. Studies on sero-prevalence and artificial infection suggested the possibility of co-infection of and reassortment between the two viruses in dogs; later, H3N1 and variants of M-variant H3N2 reassortants between pandemic H1N1/2009 and prototype H3N2 CIV were isolated. Notably, the H3N2 CIV with the matrix gene of the pdm H1N1 virus showed more efficient transmission in ferrets than the classic H3N2 CIV. These results implied that this primary companion animal, which lives in closer proximity to humans than pigs, might act as a mixing vessel or a source of novel influenza A virus in humans. Our findings emphasized the necessity of intensive monitoring for influenza infection in companion animals for investigating the potential for the emergence of novel human influenza strains.
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Species-specific restriction of Bluetongue virus replication correlates to host resilience
Bluetongue is a vector-borne disease of ruminants caused by bluetongue virus (BTV). BTV can infect essentially all domestic and wild ruminants but the clinical outcome of infection differs substantially between host species. Clinical disease induced by BTV, including haemorrhagic fever in severe cases, is normally evident only in sheep. Conversely, cattle are more resilient to BTV infection, as they develop high levels of viremia and can be reservoirs of infection, but rarely show clinical signs. Here, we concentrated on BTV-host cell interactions using primary cells as an experimental system. First, we determined that BTV reaches higher titres in ovine cells, compared to bovine cells although it induces comparable levels of antiviral cytokines in both cell types. Importantly, these differences are abolished by inhibiting the Jak/Stat pathway. In addition, pre-treatment with interferon (IFN) severely hampers BTV replication in bovine, but not in ovine, primary cells. These data suggest that bovine, unlike ovine, IFN-stimulated genes (ISGs) are effective in controlling BTV replication. Using a high-throughput flow cytometry approach, we screened an expression library of over 300 bovine ISGs to identify genes with antiviral properties against BTV. We have identified ∼10 bovine ISGs that negatively impact BTV replication (by at least 50%). Currently, we are assessing the sheep orthologues to the bovine ISGs of interest in order to investigate host-species differences. Our study provides novel insights on how bovine cells restrict BTV replication and could provide an intellectual framework to understand the host determinants involved in disease severity.
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Generation of recombinant avian coronaviruses indicates the S gene is a factor in pathogenicity
More LessThe avian coronavirus infectious bronchitis virus (IBV) is the most economically important disease of chickens in the UK, causing significant losses as a result of poor weight gain and reduced egg quality in infected birds. IBV expresses a large spike (S) glycoprotein on the surface of the virion which is responsible for attachment to host cells and is the main antigenic target for neutralising antibodies during infection. Previous work has also demonstrated that the S protein determines cell tropism in vitro. In order to investigate the involvement of the S gene in IBV pathogenesis and explore the potential for vaccine propagation in cell culture, recombinant viruses were generated using vaccinia virus based reverse genetics. Two isolates of the pathogenic M41 strain were mutated to include the S gene from a non-pathogenic lab strain with extended tropism (Beau-R) or a heterologous pathogenic field strain with restricted tropism (4/91), resulting in two recombinant IBVs termed M41K-BeauR(S) and M41K-4/91(S), respectively. These viruses were characterised in vitro and in vivo to determine the involvement of the S gene in IBV replication and pathogenicity. M41K-BeauR(S) was attenuated in vivo but exhibited the extended host tropism of the S donor strain. M41K-4/91(S) remained pathogenic and also adopted the restricted in vitro tropism of 4/91. This indicates that the S gene not only determines the cellular tropism of the virus but also plays a key role during in vivo infections, and that replacing the ectodomain of IBV S can significantly alter the pathogenicity of the resulting virus.
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Studies of cell surface and soluble HLA class I expression in pancreatic beta cells exposed to interferons or Poly I:C
More LessHLA class I (HLA-I) molecules play a crucial role in cell-mediated immunity by presenting peptide antigens to cytotoxic CD8+T cells. A pathological hallmark of Type 1 diabetes (T1D) is the hyperexpression of HLA-I in pancreatic islets that contain residual insulin producing beta cells. The expression of HLA-I can be induced following exposure to interferons (IFN). Previous studies have implicated enteroviruses (EV) as major players in triggering an autoimmune response against beta cells and it seems likely that the hyperexpression of HLA-I contributes to the recognition and targeting of beta cells by CD8+T cells. Whilst HLA-I molecules are expressed on the surface of nucleated cells, there is increasing evidence that HLA-I can also be found in a soluble form in the plasma, including in patients with viral infections. Intriguingly, soluble HLA-I (sHLA-I) levels are significantly elevated in the serum of patients with autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis and T1D, yet the function of sHLA-I is still unclear. In this project, we are investigating the impact of interferons and poly I:C, a viral dsRNA mimetic, on the expression of surface and soluble HLA-I in the human pancreatic beta cell line EndoC-βH1, the human pancreatic ductal cell line PANC1 and in HeLa cells. We show that surface HLA-I is upregulated in response to these stimuli in each of the cell lines, with greatest magnitude in EndoC-βH1. We also show for the first time that release of sHLA-I is significantly increased in response to IFNγ in EndoC-βH1 cell line.
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Proteome-wide analysis of CD8+ T cell responses to EBV lytic infection
More LessEpstein-Barr virus (EBV) is one of 9 Human herpesviruses that can develop lifelong persistence. These viruses provide an antigenically complex challenge that induce strong CD8+T cell immunity during primary infection and continue to shape this immunity through recurrent lytic reactivation. Here is described the first lytic proteome-wide analysis of CD8+T cell responses to EBV and the first to compare primary vs memory CD8+T cell responses to any human herpesvirus. Primary CD8+T cells were mitogenically expanded directly from the blood of infectious mononucleosis (IM) patients. Comparatively, memory CD8+T cells required pre-enrichment using autologous dendritic cells loaded with a lytically-infected EBV cell lysate and FACS selection based upon the activation marker 4-1BB. Enriched cells were then expanded in vitro as for IM cells. Preparations from 7 IM patients and 7 healthy carriers were screened against each of the 70 EBV lytic cycle proteins in combination with the donors’ HLA-I alleles. Multiple reactivities were identified across the full lytic cycle with 146 responses identified amongst the 7 IM patients and 96 amongst the 7 healthy carriers. However the distribution of responses varied between the 2 cohorts with primary responses targeting IE and a small group of E proteins whereas memory responses targeted all phases but with some prominent responses against L proteins. This infers that responses in primary infection therefore appear to be shaped by presentation on the infected cell surface prior to the activity of viral evasins. However long-term carriage appears to re-shape the virus-specific response.
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In vivo and ex vivo models of infectious bursal disease virus (IBDV) in inbred chicken lines differing in their resistance to the disease
More LessInfectious bursal disease virus (IBDV) targets B cells in the bursa of Fabricius (BF), causing immunosuppression in chickens and mortality. Susceptibility differs between inbred chickens, with 0 % mortality in ‘resistant’ lines and up to 80 % mortality in ‘susceptible’ lines. However, the mechanism of disease resistance is not understood. In order to address this, chickens (n=18) from three ‘resistant’ lines (15, C and O) and one ‘susceptible’ line (W) were infected with the very virulent IBDV strain, UK661. Clinical scores were recorded and tissues harvested at necropsy on day one, two and three post-infection for RNA extraction and virus titration, compared to non-infected controls. Interestingly, within a given line, we observed a range of symptoms, with some individuals experiencing more severe disease than others, despite no difference in viral replication. Line 15 was the least susceptible to disease based on the average clinical scores (3.2 (15), 5.7 (C), 4.8 (O) and 4.7 (W)) and the percentage of birds with a clinical score of 2 or above (17 % (15), 100 % (C), 83 % (O) and 83 % (W)). The average peak virus replication was also significantly lower in line 15 birds (6.3 log10 fold change) compared to lines C or O (7.0 and 6.8 log10 fold change) (P<0.01). RNA-sequence analysis will be performed using BF samples to understand the biological pathways that confer IBDV resistance. Moreover, primary bursal cells harvested from resistant and susceptible lines were infected with IBDV ex vivo and ongoing work aims to quantify differentially expressed genes in these cells.
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Unravelling a co-nsP-iracy: the role of chikungunya virus non-structural protein 3 in replication and pathogenesis
More LessChikungunya virus (CHIKV) is a member of the Alphavirus genus, transmitted to humans by mosquitoes of the Aedesgenera. Infection with CHIKV causes chikungunya fever, which in many cases can lead to chronic joint disease, leaving patients with reduced ambulation. Despite its rising potential as a threat to global health, no effective vaccine or antiviral agent for protection or treatment are available. The CHIKV non-structural protein 3 (nsP3) is essential to the virus lifecycle and is believed to be a component of the genome replication complex. However, to date, the exact role of this protein has yet been determined. Although a conserved polyproline motif in the C-terminal hypervariable domain of nsP3 has been reported to interact with cellular SH3 domains, the function of this motif remains enigmatic. To address this question we generated a panel of mutations in this motif and tested the phenotype in the context of both a subgenomic replicon and full-length infectious virus, in both mammalian and mosquito-derived cell lines. Most of the mutations were well tolerated in the sub-genomic replicon, however, a subset either attenuated or completely abolished production of infectious CHIKV. These results suggest that as well as its role in genome replication, nsP3 also functions during assembly and release of infectious virus particles and that the C-terminal polyproline motif is a critical determinant of this function.
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Modification of the ADP-ribose-1"-monophosphatase domain in recombinant infectious bronchitis virus affects viral replication in vitro and attenuates the virus in vivo
More LessThe gammacoronavirus infectious bronchitis virus (IBV) is responsible for an acute respiratory disease in domestic fowl, which has high economic impact and welfare implications in the poultry industry. The IBV non-structural protein, nsp3, is a multifunctional protein containing several putative domains, including an ADP-ribose-1’-monophosphatase (ADRP) domain conserved among coronaviruses. Inactivation of the ADRP domain in alpha- and betacoronaviruses is associated with reduced pathogenicity in vivo and altered interferon response and cytokine profiles in the host, without affecting viral replication in vitro. Therefore, recombinant viruses lacking ADRP functions have been proposed as ideal candidates for live attenuated vaccines. A recombinant IBV (rIBV) was generated in the backbone of the pathogenic M41-K strain containing a mutation in the ADRP domain catalytic core, known to abolish ADRP function in other coronaviruses. The ADRP-defective rIBV was characterised in vitro and in vivo; conversely to previously described ADRP-defective coronaviruses, in vitro analysis showed a reduction of viral replication, and the rIBV displayed a distinctive plaque phenotype. No reversion of the mutation occurred after serial passages of the virus in primary avian cell culture, nor in ex vivotracheal organ cultures which wereutilisedas a surrogate for in vivo stability testing. Pathogenicity experiments conducted in vivo resulted in a reduction in clinical signs in comparison to M41-K-infected birds, and tracheal ciliary activity, a marker for pathogenicity, was comparable to mock infected birds. These data support the role of ADRP as a pathogenic determinant and demonstrate the potential of ADRP-defective rIBV as a promising candidate vaccine.
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Role of BPIFA1 in the pathogenesis and immune response against Influenza A virus in mice
More LessThe epithelial layer of the respiratory system has a critical role in the defense against microbes and secretes a number of proteins that function in host defense. BPIFA1 is secreted by the epithelium of the respiratory tract and we have shown previously that it inhibits binding and entry of Influenza A Virus (IAV) into respiratory epithelium (Akram et al. 2018), Mucosal Immunol (11, 71). However, its precise biological functions remain unclear. The aim of this study was to assess the influence of BPIFA1 in antibody production during IAV infection. BPIFA1 KO and wild type C57BL/6J mice were infected with IAV using different virus doses and blood, broncho-alveolar lavage and nasal washes were collected at several time points for analysis of IAV-specific antibodies. The results showed that BPIFA1 has role in the efficient generation of virus-specific IgA in the respiratory tract. Thus, BPIFA1 has an important role not only in innate defense but also in the adaptive immune response against IAV.
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St. Abb’s Head phlebovirus – a separate virus species or a strain of Uukuniemi phlebovirus?
More LessSt. Abb’s Head virus (SAHV), a member of the genus Phlebovirus (family Phenuiviridae, order Bunyavirales), belongs to the largest group of negative strand RNA viruses. All phleboviruses share a genome structure that comprises three segments of negative-sense or ambi-sense RNA. The viral genome is composed of the small (S), medium (M) and large (L) RNA segments. The S segment encodes the nucleocapsid (N) protein, the M segment encodes the precursor for the viral glycoproteins (Gn and Gc) and the L segment encodes the viral RNA-dependent RNA polymerase (RdRp). Some viruses within the genus also encode non-structural proteins within their S or M segments. SAHV was isolated from a pool of seabird ticks (Ixodes uriae) collected at a seabird colony in St. Abb’s Head National Nature Reserve, Berwickshire, Scotland in 1979. Antigenically, SAHV appeared to be related to the Uukuniemi serogroup of phleboviruses. Similarly, the proteins of SAHV shared similar biochemical properties to Uukuniemi phlebovirus. Here, we describe an in depth molecular characterisation of SAHV. Using next generation sequencing technology, we demonstrate that SAHV is very closely related to the Uukuniemi phlebovirus (UUKV). We examine the growth of SAHV in mammalian, avian and tick celllines and define its target cell tropism.
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Determining the function of proteins degraded by Human Cytomegalovirus
Human Cytomegalovirus (HCMV) is associated with significant morbidity and mortality in the immunocompromised, and is a leading cause of congenital infection. Only three drugs are available for treatment, all with significant toxicities. New therapies and a vaccine are urgently required. Susceptibility to viral infection and disease is determined in part by antiviral restriction factors (ARFs) and the viral proteins that have evolved to degrade them. Small-molecule disruption of the interaction between an ARF and a viral antagonist can inhibit viral replication and may be utilised for antiviral therapies. To identify novel restriction factors against HCMV, we previously developed a multiplexed proteomic approach to identify proteins that are actively degraded early during HCMV infection. We reasoned that these would be enriched in known and novel ARFs that the virus must degrade in order to replicate. 35 proteins were shown to be degraded according to stringent statistical criteria, which included the known anti-HCMV restriction factors Sp100 and MORC3, and a novel ARF, HLTF. Here, we present preliminary results from a combination of viral replication assays to identify other novel ARFs. These include a new ‘two-colour’ approach to characterise HCMV restriction, which aims to eliminate variability in cell density by mixing populations of cells in a single cell culture well. Preliminary studies have identified a number of proteins that inhibit virus replication, as well as a novel dependency factor which the virus may degrade in order to attenuate cellular immune signalling or help establish latency.
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Evaluating the epizootic risk to poultry of a novel Chinese H7N9 virus variant with increased pathogenicity in turkeys
Previously we successful infected turkeys with the China-origin H7N9 low pathogenicity avian influenza virus (LPAIV, A/Anhui/1/13, referred to as ‘wild-type’ (Wt)) which successfully transmitted to contact turkeys with virulent outcomes, highly unusual for LPAIV infection, particularly as the LPAIV cleavage site remained unchanged in all experiments. Sequencing of progeny viruses revealed consistent emergence of the L226Q polymorphism in the HA gene, termed the ‘turkey-adapted’ (ty-ad) virus. Ty-ad and Wt were used to compare the epizootic risk posed by both H7N9 LPAIVs in turkeys and to explore the mechanisms which underpins any differences. The Wt and ty-ad viruses robustly infected inoculated and contact turkeys, producing similar shedding titres. However, the ty-ad virus was more pathogenic than the Wt virus in directly-infected and contact turkeys, causing 100 % (Wt) compared to 16 % (ty-ad) survival. The ty-ad virus was detected in broader range of turkey organs, and at higher titre, compared to the Wt variant. This contrasted with pathogenicity and tissue-tropism observations for both viruses in chickens. The wt and ty-ad viruses did not replicate without trypsin in vitro, affirming a typical LPAIV phenotype. The L226Q polymorphism is known to alter receptor binding, with key differences in receptor distribution between turkey and chicken tissues observed. Replication kinetics differences in a range of avian cells will be reported for both viruses. Consequently, if this ty-ad variant were to arise more frequently in nature, it would pose an increased virulent risk to turkeys. It is therefore important to maintain surveillance and understanding of China-origin H7N9 viruses.
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African Horse Sickness virus: pathogenicity in an IFNAR(-/-) mouse model of infection
More LessAfrican Horse Sickness (AHS) is a highly lethal, vector-borne viral disease of equids, endemic to sub-Saharan Africa but with a history of outbreaks into Europe. The pathogenesis of the virus is not fully understood; some studies have shown that certain proteins are linked to virulence, and that the outcome of infection is highly dependent on viral factors. Disease can also manifest with several different presentations in the equine host (fever form, cardiac form, pulmonary form and mixed form) but the mechanism underlying this variation is not fully understood. Pathogenicity and virulence studies of AHSV are difficult to perform in horses for logistical, ethical and financial reasons. As an alternative, Interferon-alpha receptor knockout (IFNAR-/-) mice have previously been used in AHSV vaccinology studies. Full pathology characterization of the AHSV infection in this model is the primary objective of our work, which will address questions regarding pathogenesis of AHSV. Here we present data collected from experimental infection of IFNAR (-/-) mice with a strain of AHSV serotype 4, including: clinical signs; histopathology; immuno-histochemical analysis of immune response to infection; antigen detection in tissues; and transmission electron microscopy of AHSV infected tissues. In addition, we will show data from experimental infection in this animal model comparing the pathogenicity of different AHSV strains. Results obtained indicate AHSV-4 infection is correlated with oedema and pneumonia in the lungs, inflammation in the liver and meningitis plus perivascular cuffing in the cerebrum. Other data shows that different strains of AHSV differ in terms of their pathogenicity and tropism.
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Characterising the insect-borne transmission of the cattle poxvirus lumpy skin disease virus
Lumpy skin disease (LSD) is an emerging poxviral disease of cattle caused by the Capripoxvirus lumpy skin disease virus (LSDV) which generates widespread cutaneous lesions in affected animals. LSD is recognised as a transboundary high consequence disease in Africa where it contributes to rural poverty and food insecurity. In 2015 LSDV spread to southeastern Europe and currently poses a threat to cattle in neighbouring regions. Previous research indicates that LSDV is most likely transmitted by insect vectors however details of transmission pathways are unclear. This study was designed to identify the risk of transmission of LSDV posed by different insect vectors. A bovine experimental model of LSD was established in the high containment facilities at The Pirbright Institute. Potential insect vectors (Aedes aegypti, Culex quinquefasciatus, Stomoxys calcitrans and Culicoides nubeculosus) were fed on LSDV-infected cattle then incubated for up to eight days. Cattle and insects were regularly sampled to quantify LSDV present in different tissues and vector species. This data was then used to model the dynamics of LSDV infection and transmission. All four species of vector successfully acquired LSDV from infected cattle and maintained the virus up to eight days post feeding. The outputs of this research will now be used to design more effective LSD control programmes.
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Swine influenza A viruses with zoonotic potential – PCR HA/NA typing, and differential detection of pandemic09 reassortants in GB and European pigs
Swine influenza A virus (swIAV) causes respiratory disease and productivity loss in pigs. Swine ‘flu viruses have been known to be both zoonotic and reverse zoonotic and they contain genes of swine, avian(av) and human(hu) origin. Surveillance of swIAV subtypes is important as genotypes/phenotypes are fluid and impact with respect to epidemiology, vaccination, pig welfare, veterinary and public health. Three sub-types (H1avN1, H1N1pdm09, H1huN2) are currently found in pigs from Great Britain (GB), plus H3huN2 in Europe and their reassortants. Screening of candidate samples is carried out by RRT-PCR assays – generic detection of swIAV (M gene) followed by a specific RRT-PCR for H1N1pdm09 (HA gene), a suite of RRT-PCR assays for sub-typing (HA and NA genes) and a (differential) RRT-PCR to specifically identify reassortant swIAVs that incorporate the pandemic 2009 internal gene cassette (NP gene). Subtyping assays, conventional and/or molecular, are carried out on virus isolation-positive and –negative (RNA only) samples from clinical material (respiratory tissue and/or nasal swabs). Since 2009, the number of swIAV has expanded with the H1N1pdm09 isolates reassorting with the traditional subtypes. Many European variants arose (>25) of which some have become established – in GB including H1huN2/pdm (since 2010), and H1avN1/pdm (since 2012), and in Belgium the traditional isolates were detected plus H1pdmN1/pdm and H3huN2/pdm reassortants. PCR subtyping (2012 onwards ∼130 from GB and ∼40 from BE/NL), wholegenome sequencing and bioinformatics analysis of these isolates facilitate further diagnostic improvements and assessment of zoonotic pandemic potential (in silico and in vivo).
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Replicative fitness and transmission of G57 lineage and UDL01 like H9N2 viruses in chickens
More LessThe low pathogenic H9N2 influenza viruses are a threat to poultry as well as global public health due to their ability to reassort with other avian influenza viruses leading to the emergence of novel reassortant viruses having pandemic potential. The continued inter-subtypic reassortment events between influenza viruses in the Indian sub-continent have led to the replacement of the already existing G1 lineage of H9N2 viruses with the UDL genotype-like (A/chicken/Pakistan UDL-01/08/H9N2) viruses, which are triple reassortants between H9N2 virus (G1 lineage), HPAI H5N1 virus (clade 2.2) and HPAI H7N3 viruses. G1 lineage of H9N2 viruses in China has also been replaced with a fitter G57 lineage which donated internal genes to novel H7N9 viruses in 2013. We assessed and compared the replication, transmission and pathogenic potential of UDL01/2008/H9N2 virus and A/Ck/Vietnam/H7F-14-BN4-315/2015 H9N2 virus of G57 lineage isolated from Vietnam in 2015. Vietnam H9N2 virus was found to be relatively more virulent compared to the UDL genotype-like H9N2 in Chickens. Our in-vitro and in-ovo infection studies also showed that Vietnam/BN4-315/H9N2 virus has greater replication fitness compared to UDL-01/08/H9N2 virus. The UDL-01/08 H9N2 reassortants carrying internal genes of Vietnam/BN4-315 virus also showed improved replication fitness in MDCK cells. It is, therefore predicted that genetic reassortment between dominant strains in the Far East and the Indian subcontinent/Middle East may generate more virulent H9N2 viruses.
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