- Volume 1, Issue 1A, 2019
Volume 1, Issue 1A, 2019
- Oral Abstract
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- Virology Workshop: Morphogenesis, Egress and Entry
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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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Prevalence and resistance pattern of uropathogens from community settings of different regions: an experience from India
Sarita Mohapatra, Rajashree Panigrahy, Vibhor Tak, Shwetha J. V., Sneha K. C., Susmita Chaudhuri, Swati Pundir, Deepak Kocher, Hitender Gautam, Seema Sood, Bimal Kumar Das, Arti Kapil, Pankaj Hari, Arvind Kumar, Rajesh Kumari, Mani Kalaivani, Ambica R., Harshal Ramesh Salve, Sumit Malhotra and Shashi Kant
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