- Volume 1, Issue 1A, 2019

Asthma affects over a million children in the UK. Early age viral infection, allergen sensitization, atopy and genetic predisposition increases the likelihood of developing asthma. Our aim is to understand the consequences of allergen exposure (house-dust mite; HDM) and/or RSV infection on innate immune responses, cytopathogenesis and virus replication in paediatric airway epithelium. Well-differentiated primary paediatric nasal epithelial cell (WD-PNEC) cultures derived from newborn or pre-school children were infected with RSV before or after stimulation with HDM. Virus growth kinetics, cytopathology and innate immune responses were analysed. HDM pre-treatment did not affect the culture integrity or RSV growth. Preliminary RT-qPCR analysis revealed upregulation of irf9 in RSV infected/HDM treated and HDM pre-treated/RSV infected cultures at 24 and 96 h post-infection (hpi). Isg15 was also upregulated in HDM pre-treated/RSV infected cultures at 24 hpi in newborns only. At 96 hpi, irf9, isg15, ifi6, duox2 and duoxA2 were upregulated in RSV infected cultures. Surprisingly, HDM treatment alone did not induce any significant upregulation of these genes compare to untreated/uninfected controls. However, data from more donors will be required to confirm these preliminary data. IL-29/IFNλ1 secretion was also significantly reduced following HDM pre-treatment of RSV infected cultures, while there was a trend towards reduced IL-6 secretion. These results suggest that HDM exposure modulates the innate immune responses to RSV infection of airway epithelium. The preliminary data are consistent with our over-arching hypothesis that our model of aeroallergen exposure and viral infection of airway epithelium will help elucidate mechanisms by which pre-school wheeze develops.

Vaccinia virus (VACV) encodes multiple proteins that function to inhibit both the production and downstream effects of interferons, which are critical modulators of the antiviral response. One such protein, VACV C6, inhibits both the production of type I IFN (by blocking IRF3 signalling) and the downstream effects of type I IFN (by blocking JAK-STAT signalling). Further to this, our findings suggest C6 can also inhibit the downstream effects of type II IFN of which IFN-gamma is the sole member. The observation that C6 co-immunoprecipitates with STAT1, a protein crucial in activating the downstream functions of IFN-gamma, might explain its inhibitory activity.

Influenza A virus (IAV) is a major causative agent of respiratory tract infection in humans. Human IAVs cause seasonal epidemics, whilst avian IAV strains sporadically cross the host range barrier leading to zoonotic infections. The complement system is a component of the innate immune system that acts to remove pathogens including IAVs. Many bacterial pathogens and several viral pathogens have been reported to actively target components of the complement activation pathway as a defensive strategy. The aim of this project is to understand if there is an interaction between IAV and co-factor H, a critical inhibitor of the complement system and what effect any interaction may have on influenza replication. Using purified human co-factor H protein and purified human (H1N1 and H3N2) and avian (H9N2 and H5N3) IAVs we have demonstrated using ELISA assays that an interaction does occur between co-factor H and all the IAV strains tested. Far western blot analysis suggests that the interaction is with the viral Hemagglutinin (HA), which is responsible for attachment and entry of IAV to cells. Interestingly the interaction has divergent effects on the replication of the IAV strains; enhancing human H1N1 replication and restricting H3N2 replication in A549 and THP-1 cells whilst having no effect on the replication of avian H5N3 or H9N2 viruses. Understanding IAV interaction with the complement pathway could enhance our ability to produce effective vaccines.

Respiratory syncytial virus (RSV) infection is the major cause of severe lower respiratory tract disease in young infants. Evidence suggests that cystic fibrosis (CF) is associated with significant morbidity following RSV infection. We and others previously demonstrated that airway epithelium is the primary target of RSV infection. However, little is known about the impact of RSV infection on CF airway epithelium. To address this, we established and characterised well-differentiated primary nasal epithelial cell cultures (WD-PNECs) from recently diagnosed CF infants to study RSV cytopathogenesis in CF airway epithelium. CF WD-PNECs were successfully generated from 11 infants and characterised by light and fluorescent microscopy. CF WD-PNECs secreted thick dry apical mucous, consistent with in vivo observations. Extensive cilia coverage was also evident, although cilia beat frequencies appeared lower than those evident in WD-PNECs from healthy neonates. Quantification of goblet and ciliated cells in the CF WD-PNEC cultures were similar to those of healthy cultures. Viral growth kinetics were similar for CF WD-PNECs and healthy WD- PNECs, with peak virus titres evident at 72–96 hpi. CXCL8/IL-8 and CXCL10/IP-10 secretions were upregulated following RSV infection of CF WD-PNECs, while IL-6 secretions did not change. Interestingly, our data suggested that duoxa2 and duox2 expression post-infection were reduced in WD-PNECs from CF compared to healthy infants. Our data suggest that this model provides an exciting opportunity to elucidate the cytopathogenic, inflammatory and molecular consequences of RSV infection of airway epithelium derived from very young CF infants.

Harry Smith Vacation Studentship Laboratory-adapted influenza viruses produce predominantly spherical virions. In contrast, clinical and veterinary isolates produce a mixture of virions of different sizes, from 0.1 µm spheres to filaments which can reach tens of microns in length. Filamentous influenza virions were discovered in 1946, but the bulk of influenza research has analysed only spherical forms of the virus and the role of filaments in influenza infections is unclear. Functional studies of filaments require the development of methods to manipulate the ratio of spherical to filamentous virions, and we reasoned that this could be achieved by filtration. To test this, we infected MDCK cells with the filamentous Udorn strain of influenza A virus. We collected virus-containing growth media and passed this through filters with 5 µm, 0.45 µm and 0.2 µm pores. Filtrates and unfiltered virus were compared, using Western blotting to measure their protein composition, plaque assays to measure their infectivity and negative stain transmission electron microscopy to measure individual particle sizes. We found that filtration through a filter with 5 µm pores had little effect on composition, infectivity and the ratio of spherical to filamentous particles. In contrast, sub-micron filters, particularly those with 0.2 µm pores, caused a general depletion of virions but increased the sphere to filament ratio. We therefore concluded that sub-micron pore sizes can be used to preferentially remove filaments from populations of pleomorphic influenza virions, providing a useful tool for subtractive studies of the contribution filaments make to influenza virus infections.

Most laboratory strains of influenza virus produce near-spherical virions, but clinical isolates also produce extended filaments whose biophysical properties are understudied. Most functional studies of filamentous influenza viruses do not include data on the concentration or lengths of the virions, making it hard to interpret their sometimes contradictory results. Furthermore, anecdotal reports suggest that filaments are damaged during routine laboratory handling. Therefore, to understand filament function we require a tool to assess the number and dimensions of filaments in a sample and an assessment of how filaments respond to standard handling procedures. We initially sought to analyse filament populations using negative stain particle counting, but found that this was low-throughput and could not detect particles longer than 10 µm. Instead, we used confocal microscopy with semi-automated image analysis. This allowed a high-throughput, quantitative analysis of length distributions in filament populations. Using this, we assessed the effects of pipetting, vortexing, sonicating, clarification and freezing on filaments. Most procedures did not appreciably alter filament dimensions. Pipetting and vortexing both slightly reduced filament numbers, but their effects were only appreciable after extended treatment. In contrast, freezing substantially reduced the number and median length of filaments, as well as creating ‘kinks’ in filaments which suggest damage to the capsid. We conclude that confocal microscopy can provide the basic measurements needed to interpret functional studies of filamentous strains. Using this approach, we found that freezing filaments causes previously unappreciated damage, which should be considered when planning further research.

There are seven ‘established’ morbilliviruses: rinderpest (RPV), measles (MeV), small ruminant (PPRV), cetacean (CeMV), phocine (PDV), canine (CDV) and feline (FmoV) morbillivirus, the majority of which are thought to have narrow host ranges. The exception is CDV, which has been reported in almost all families of the order Carnivora, as well as non-human primates. Morbilliviruses enter cells through one of two proteinaceous receptors: signalling lymphocyte activation molecule F1 (SLAMF1), present on the surface of many immune cells; or Nectin-4, expressed on the basolateral side of various polarised epithelial cells. Given the close similarity of these viruses and their universal use of related receptors, there are realistic fears morbilliviruses may spill-over into naïve hosts, i.e. following eradication of RPV in cattle. This spill-over is not entirely unprecedented; PDV and MeV are thought to share common ancestors with CDV and RPV, respectively. To examine morbillivirus receptor usage and predict future spill-over and zoonotic transmission events, we have developed a high-throughput, quantifiable, cell-cell fusion assay. Effector cells, expressing the viral F and H glycoproteins as well as a split-luciferase reporter, are co-cultured with target cells expressing SLAMF1 or Nectin-4 receptors and the remaining half of the reporter. Upon functional receptor engagement the effector and target cells fuse, allowing cytoplasmic mixing and reconstitution of the reporter, a process which can be quantified. Using these assays we are investigating whether the receptor usage profile of morbilliviruses represents an important factor in spill-over transmission into new hosts.

Herpes Simplex Virus (HSV) afflicts ∼90 % of the population, causing a range of diseases from oral cold sores to encephalitis. Despite the widespread effects of HSV, current drugs cannot cure infection and there are no preventative vaccines. HSV infection requires entry into the host cell, which is mediated by the fusion protein, gB. Despite various structures being available for post-fusion gB, a high-resolution model for the pre-fusion state, required to understand the fundamental mechanism of HSV fusion, is still lacking. To elucidate this structure, full length gB was previously expressed on lipid vesicles and was shown via cryo-electron microscopy techniques to adopt the elusive pre-fusion state. Strikingly, two recent studies, one characterising such vesicles, generated two different pre-fusion gB models. The discrepancy between the models can be summarised by the position of the fusion loops: are they positioned facing away from the viral membrane or towards it in the pre-fusion state? Using the gB studded vesicle production method combined with a state-of-the-art Titan Krios cryo-electron microscope equipped with a Volta Phase Plate, a direct electron detector camera and an energy filter, all to increase contrast and signal-to-noise ratio, we aim to settle the dispute as to the structure of pre-fusion gB. Revealing the pre-fusion structure could lead to possible therapeutics to a disease that can have serious complications in newborns and the immunocompromised, a growing cohort in the modern world.

Viral glycoproteins are found on the surface of all enveloped viruses, mediating binding to host receptors and the initiation of entry events. In addition, numerous vaccines employ the same viral glycoproteins as immunogens, either vectored or recombinant in nature. During infection viral glycoproteins are thought to interact with various host-factors, facilitating their trafficking to the cell surface. However, these interactions are not currently well understood or characterised. Using a human gene expression microarray, the cellular response to expression of various viral glycoproteins (Ebola, Nipah, VSV and Measles) was assessed in vitro. Specifically, we found a number of genes with a fold change greater than 2 displaying significantly altered expression across all four glycoprotein transfections. A subset of these genes were selected for validation by qPCR and extended to RSV (respiratory syncytial virus) fusion protein (F) transfection. The expression of these genes was then further investigated in Measles and RSV infected cells. Our data has identified host genes with altered expression in response to a diverse panel of viral glycoproteins, potentially elucidating a conserved set of pathways important for viral glycoprotein activity. Greater understanding of the proteins and pathways involved in glycoprotein expression has the potential to identify mechanisms underpinning host susceptibility to disease as well as improving the yield of vaccine producing cells.

Equine hepacivirus A (EqHV) belongs to the family Flaviviridae and has been identified as the hepacivirus phylogenetically most closely related to Hepatitis C virus (HCV). Like HCV, EqHV is a hepatotropic virus that has been reported in over fourteen countries from six continents. It has a high prevalence in some populations, in particular the Thoroughbred breed. However, much is still unknown about EqHV, such as its pathogenesis and mode of transmission. The main four receptors used by HCV for viral entry to human hepatocytes are Cluster of Differentiation-81 (CD-81), occludin (OCLN), claudin-1 (CLDN-1) and Scavenger Receptor Class B Member 1 (SCAR-B1). This study investigated the distribution and expression of these entry receptors on equine cells by flow cytometry, immunocytochemistry and immunohistochemistry. Using a human liver cell line (Huh-7) as a positive control, antibodies against HCV receptors appeared to cross-react with antigens on equine cells. The proportion of fetal horse kidney cells that expressed CD-81, OCLN, and CLDN-1 was 37.2 %, 16.0 %, and 7.0 %, respectively, whereas the equine dermal cells (E.Derms) expressed CD-81 (96.0 %), OCLN (1.2 %) and CLDN-1 (1.7 %). CD-81, OCLN and CLDN-1 were also expressed on E.Derms and Huh7 cells, as detected by immunocytochemistry and on equine liver cells and the allantochorionic region of Thoroughbred placenta by immunohistochemistry. These findings form the basis for further comparative investigation into the entry receptors used by EqHV to infect equine and human cells. Such information may inform future studies on EqHV pathogenesis and mode(s) of transmission.

Respiratory syncytial virus (RSV) is the major cause of childhood respiratory disease; however, currently there is no licenced vaccine available and the only therapeutic, a monoclonal antibody against the viral Fusion (F) protein, is expensive and applied sparingly. RSV particles enter cells by membrane fusion, orchestrated by F – a type I integral membrane protein. This process was recently shown to involve macropinocytosis of the particle. Separately, RSV can spread through induction of direct cell-cell fusion – again orchestrated by F. Little is currently known about the host-factors involved in regulating or inhibiting RSV F-mediated fusion. Here, using two different high-throughput screening approaches, we have identified host-factors involved in regulating RSV fusion. Using quantitative mass-spectrometry analysis of isolated cell membrane fractions from mock and RSV-infected cells we have identified membrane proteins which are differentially regulated during RSV infection. Furthermore, using lentiviral libraries expressing individual interferon stimulated genes (ISGs) from different mammalian species we have investigated ISG-mediated inhibition of RSV fusion. Our data provides important insights into host-factors involved in RSV spread, furthering our understanding of the fusion process and identifying potential targets for antiviral therapy.

The herpesvirus family contains eight human pathogens that act as the causative agent of several human maladies. The herpesvirus family can be further subdivided alphaherpesviruses, betaherpesviruses, and the oncogenic gammaherpesviruses. Herpesvirus replication and capsid assembly occurs within the nucleus, from there capsids migrate into the cytoplasm. Following nuclear egress, viral capsids travel to the cellular membrane, where morphogenesis takes place. Recent analysis has demonstrated that the gammaherpesvirus Kaposi’s sarcoma associated herpesvirus (KSHV) upregulates the expression of a host miR-365 to target the cellular protein, DOCK5, to enhance KSHV egress. This hypothesis is supported by the observation that inhibition of miR-365 or overexpression of DOCK5 leads to the prevention of KSHV egress and accumulation of capsids at the plasma membrane. As herpesviruses share several mechanistic features, we are now employing electron microscopy to visualise the cytoplasmic capsid accumulation and to determine how DOCK5 is involved in herpesvirus egress. This will be achieved through advanced microscopy techniques such as correlative light microscopy, cryo-electron microscopy, and a novel nanobiopsy approach known as nanopipetting. Currently we have been establishing the optimal time of capsid egress in both KSHV and Herpes Simplex Virus-1 which will aid in visualising the process through transmission electron microscopy. A better understanding of herpesvirus egress will potentially help identify novel antiviral targets against herpesviruses.

While influenza A and B virus contain the two glycoproteins Hemagglutinin (HA) and Neuraminidase (NA) inserted into the viral membrane, influenza C virus possesses only one spike designated Hemagglutinin-Esterase-Fusion (HEF) protein which combines the functions of both HA and NA. Like HA, it recognizes and binds to a receptor on the cell surface to initiate virus entry. However, the receptor is N-acetyl-9-O-acetylneuraminic acid. HEF is the receptor-destroying enzyme, which is the function of the neuraminidase (NA) in influenza A and B virus. Although Influenza C virus infections are generally mild and self-limited, it comprises around 13 % of influenza positive cases in hospitalized children. It is also a cause of community acquired pneumonia of children. As influenza research has been more focused on Influenza A and B, it is of interest to pseudotype influenza C HEF. The full sequence of Inflenza C HEF (C/Tokyo/4/2014) was obtained and cloned into pi.18 expression plasmid. The second generation packaging construct pCMVΔR8.91, and the self-inactivating lentiviral vector pCSFLW expressing firefly luciferase, and pCSGW expressing GFP were used for lentiviral vector particle production. The original HEF sequence was mutated by site directed mutagenesis to inhibit esterase activity to prevent receptor destruction prior to pseudotype viral entry. Pseudotyped particles bearing the HEF glycoprotein were successfully produced and verified by fluorescence and Luciferase titration assay with titres of 5×107 RLU/ml. Currently, optimization is undergoing to achieve higher titres as well determining the specific protease/s utilized by HEF for its activation.

More than 50 % of cattle (regardless of foot and mouth disease (FMD) vaccination) become persistently infected for long periods of time (years) after exposure to FMD virus (FMDV). The mechanisms associated with establishment of persistent infections are still poorly understood. I am testing the hypothesis that complement receptor 2 (CR2) on follicular dendritic cells (FDCs) located in the germinal centers of the lymphoid tissue, are involved in the trapping and long-term persistence of FMDV and that this persistence is essential for the maintenance of long-term antibody responses to FMDV. The aim of this study was to assess FMDV antigen retention and the generation of the specific immune response in vivo, in a mouse model. Groups of mice were treated with an anti-CR2 monoclonal antibody, named 4B2, which has been described previously to block CR2 long-term in vivo, blocking for 6 weeks after a single injection of 2 mg (Kulik et al., 2015). After treatment with 4B2, animals were infected with FMDV and lymphoid tissues and serum samples evaluated for the presence of antigen and the humoral immune response, respectively. The ability of 4B2 to block the binding of FMDV and PAP immune complexes (ICs) to FDCs in vitro as well as results about the role of FDC in trapping FMDV via CR2 in vivo is currently being investigated and will be discussed.

Various influenza A viruses (AIVs) have infected humans in the last decade. These infections result from spill over into humans at times of AIV poultry outbreaks with severe clinical consequences for those infected. These AIVs do not have the ability to be efficiently spread among humans via respiratory droplets or aerosols and subsequently cause a pandemic. However, adaptation of these avian influenza viruses to humans by mutation or reassortment may change this. We know that AIVs require adaptation of several critical characteristics in order to effectively spread between humans. Therefore, in order for human pandemic emergence, AIV that crosses into a human host must already be sufficiently able, or must rapidly adapt to overcome these constraints to transmit to a subsequent human host before infection is cleared by an immune response. In this study, we used mice and analysed the mutations acquired by avian origin H7N9 and H9N2 isolates in a temporal fashion during a single infection cycle in individual hosts. Viruses recovered from infected mice lungs were subjected to Next Generation sequencing (NGS) to identify mammalian adaptation enhancing mutations. Moreover, the timing of these mutations was correlated to the host response, with particular emphasis on cytokine profile and innate immune responses during the time prior to and following viral sequence changes to help understand the virus-host dynamics and the host environment favouring adaptation. Furthermore, understanding of the determinants and mechanisms of adaptation and transmission may aid in assessing the risks posed by avian influenza A viruses to human health.

Influenza D virus (IDV) is a newly described member of the Orthomyxoviridae family, initially identified during a 2011 outbreak of respiratory disease in North American pigs. Cattle were subsequently shown to be the main reservoir of the virus and accumulating evidence suggests a role for IDV in bovine respiratory disease complex. During the winter of 2017/2018, cattle submitted to the Agri-Food and Biosciences Institute for post-mortem with confirmed respiratory disease were tested for the presence of IDV by real-time RT-PCR. Virus isolation was performed in Swine Testes cells and full-genome sequence determined. Of 104 cattle with confirmed respiratory disease, 9 tested positive for IDV (8.7 % prevalence). Virus was detected in both the upper and lower respiratory tract. Lung tissues from IDV positive samples were negative for the presence of bovine herpesvirus 1, bovine respiratory syncytial virus, bovine viral diarrhea virus and parainfluenza virus 3. Of the 9 cattle which tested positive for IDV, 3 tested positive for coronavirus. Histological analysis of lungs from IDV positive samples revealed pathological features including necrosis, neutrophil infiltration of alveolar spaces, fibrosis, congestion, oedema and haemorrhage. Sequenced isolates were shown to cluster with European isolates of the D/swine/Oklahoma/1334/2011 clade. To date, IDV has been detected in North America, Mexico, Japan, China, France, Italy and the Republic of Ireland. This study is the first to identify IDV in UK cattle herds. The presence of IDV in respiratory disease samples supports a role for this virus in bovine respiratory disease complex.

Bluetongue virus (BTV) is an arbovirus transmitted by biting midges (Culicoides pp.). BTV causes a severe disease (bluetongue) in domestic and wild ruminant species with high levels of morbidity and mortality. Bluetongue has emerged as an important disease in sheep and cattle worldwide. The BTV genome is composed by ten linear dsRNA segments, packaged within a triple-layered icosahedral protein-capsid, and encode 7 structural and 4/5 non-structural proteins. To date, there are at least 27 BTV serotypes (mainly determined by the VP2 outer capsid protein) circulating worldwide. In addition, high rates of reassortment involving all genome segments have been documented, complicating epidemiological studies and vaccination programmes. We have developed BTV-GLUE (http://btv.glue.cvr.ac.uk), a new bioinformatics sequence data resource for bluetongue virus. Sequences from the NCBI nucleotide database are curated along with complementary sequence metadata and are integrated together inside GLUE (http://tools.glue.cvr.ac.uk), a data-centric software package for capturing virus sequence data and organising it along evolutionary lines. The dataset also contains reference sequences with genome feature annotations, multiple sequence alignments, defined clades and phylogenetic trees, for each BTV segment and clade. A new automated genotyping tool for all segments has been developed. The resource may also be used as an offline bioinformatics toolkit. BTV-GLUE will help the BTV community to study varying aspects of BTV biology and evolution and will facilitate the adoption of a nomenclature that more easily distinguishes the properties of BTV strains circulating worldwide.

Zika virus (ZIKV) was originally described during 1947 in Uganda when it was isolated from a sentinel macaque in the forest of Zika. After its discovery, discrete outbreaks were reported in Africa and Asia, but it was until 2007 when an outbreak in the Yap Islands where it was identified as a pathogen capable of causing epidemics and became associated with microcephaly. We analysed 10 ZIKV sequences from key locations, tracking the virus’ spread through the Americas, following the Yap Island outbreak. We identified 8 mutations within the M (prM) and E proteins which may alter the tropism or entry kinetics of the virus. To test the influence of these glycoprotein mutants on virus entry we have explored lentivirus pseudotypes with a luciferase reporter to measure infectivity. So far, the model involving the use of PNL 4.3 HIV based retroviral backbone has not given favourable results so in the aim to try to improve the conditions and favour the process we tested different conditions including a matrix of concentrations between the retroviral backbone and the viral glycoprotein. The addition of other viral proteins was also tested in an effort to produce particles capable to infect the target cells.