Feline Stem Cell Factor: Isolation and Characterisation of Biological Activity

Cytokines are small proteins produced by many tissue types and have wide ranging effects on the haemopoietic and immune systems. The cloning of human cytokines has facilitated the production of recombinant cytokines in quantities sufficient to enable detailed study of their biological properties. An understanding of the biological effects of these cytokines has led to their introduction as novel therapeutic agents with widespread potential uses, including the treatment of cancer, cytopenias and viral infections. The use of heterologous cytokines in domestic species has been of only limited success, in part due to the variable degree of interspecies conservation. In order to fully realise the potential for cytokines as therapeutic agents and to facilitate further studies of the role of cytokines in diseases of domestic species, the isolation of species specific cytokines is desirable. This thesis describes the approach used to isolate and clone feline stem cell factor (fSCF) and subsequently express the recombinant protein and characterise its biological properties. Stem cell factor is the ligand for the tyrosine kinase receptor encoded by the c-kit gene. It has wide ranging actions on cells of the haemopoietic, reproductive and nervous systems and melanocytes, in particular promoting the survival and development of primitive cells. cDNA clones encoding two isoforms of fSCF were isolated using RT-PCR and their sequences determined. The cDNAs encode a predicted full length fSCF protein of 274 amino-acids and a shorter isoform of 246 amino acids. Feline SCF shows a high degree of homology to the SCFs of other species at both the nucleic acid and protein level. Feline SCF was expressed as a soluble protein using the glutathione S-transferase fusion protein system and purified by affinity, anion exchange and gel filtration chromatography. Murine MC/9 and human TF-1 cells were used to assay fSCF biological activity. The recombinant protein supported the growth of feline granulocyte-macrophage colony forming cells in vitro and in combination with feline phytohaemagglutinin lymphocyte conditioned medium increased colony numbers and sizes were seen. Administration of the recombinant protein to cats produced increases in circulating colony forming cells, induced extramedullary haemopoiesis in the spleens of treated cats and led to increased mast cell numbers at the site of injection. In order to enable assessment of the effects of frSCF upon primitive haemopoietic cells, the production of polyclonal antiserum to CD34 (a transmembrane glycoprotein expressed predominantly on primitive haemopoietic cells) was attempted. Rabbits were used to raise antisera to conserved intracellular epitopes of the CD34 molecule by inoculation with immunogenic peptides. This was of limited success; whilst the antisera recognised the synthetic peptides against which they had been raised, they showed poor affinity for the native protein. These studies provide the basis for further investigations of the potential of this cytokine in the treatment of feline disease, particularly cytopenias associated with neoplasia, chemotherapy or viral disease (e.g. FeLV, FIV) and in the development of peripheral stem cell transplantation. The ability of fSCF to synergise with other cytokines in vitro suggests that it may be combined with other haemopoietic cytokines in vivo to provide more potent haemopoietic stimulation. Furthermore, the recombinant cytokine may be usefully employed to support in vitro growth of haemopoietic cells in this species and so facilitate their study

A consensus approach to vertebrate de novo transcriptome assembly from RNA-seq data: assembly of the duck (Anas platyrhynchos) transcriptome

For vertebrate organisms where a reference genome is not available, de novo transcriptome assembly enables a cost effective insight into the identification of tissue specific or differentially expressed genes and variation of the coding part of the genome. However, since there are a number of different tools and parameters that can be used to reconstruct transcripts, it is difficult to determine an optimal method. Here we suggest a pipeline based on (1) assessing the performance of three different assembly tools (2) using both single and multiple k-mer (MK) approaches (3) examining the influence of the number of reads used in the assembly (4) merging assemblies from different tools. We use an example dataset from the vertebrate Anas platyrhynchos domestica (Pekin duck). We find that taking a subset of data enables a robust assembly to be produced by multiple methods without the need for very high memory capacity. The use of reads mapped back to transcripts (RMBT) and CEGMA (Core Eukaryotic Genes Mapping Approach) provides useful metrics to determine the completeness of assembly obtained. For this dataset the use of MK in the assembly generated a more complete assembly as measured by greater number of RMBT and CEGMA score. Merged single k-mer assemblies are generally smaller but consist of longer transcripts, suggesting an assembly consisting of fewer fragmented transcripts. We suggest that the use of a subset of reads during assembly allows the relatively rapid investigation of assembly characteristics and can guide the user to the most appropriate transcriptome for particular downstream use. Transcriptomes generated by the compared assembly methods and the final merged assembly are freely available for download at http://dx.doi.org/10.6084/m9.figshare.1032613

A consensus approach to vertebrate de novo transcriptome assembly from RNA-seq data: assembly of the duck (Anas platyrhynchos) transcriptome

For vertebrate organisms where a reference genome is not available, de novo transcriptome assembly enables a cost effective insight into the identification of tissue specific or differentially expressed genes and variation of the coding part of the genome. However, since there are a number of different tools and parameters that can be used to reconstruct transcripts, it is difficult to determine an optimal method. Here we suggest a pipeline based on (1) assessing the performance of three different assembly tools (2) using both single and multiple k -mer (MK) approaches (3) examining the influence of the number of reads used in the assembly (4) merging assemblies from different tools. We use an example dataset from the vertebrate Anas platyrhynchos domestica (Pekin duck). We find that taking a subset of data enables a robust assembly to be produced by multiple methods without the need for very high memory capacity. The use of reads mapped back to transcripts (RMBT) and CEGMA (Core Eukaryotic Genes Mapping Approach) provides useful metrics to determine the completeness of assembly obtained. For this dataset the use of MK in the assembly generated a more complete assembly as measured by greater number of RMBT and CEGMA score. Merged single k -mer assemblies are generally smaller but consist of longer transcripts, suggesting an assembly consisting of fewer fragmented transcripts. We suggest that the use of a subset of reads during assembly allows the relatively rapid investigation of assembly characteristics and can guide the user to the most appropriate transcriptome for particular downstream use. Transcriptomes generated by the compared assembly methods and the final merged assembly are freely available for download at http://dx.doi.org/10.6084/m9.figshare.1032613. © 2014 Moreton, Dunham and Emes

Lipid biophysics and/or soft matter-inspired approach for controlling enveloped virus infectivity

Proven as a natural barrier against viral infection, pulmonary surfactant phospholipids have a biophysical and immunological role within the respiratory system, acting against microorganisms including viruses. Enveloped viruses have, in common, an outer bilayer membrane that forms the underlying structure for viral membrane proteins to function in an optimal way to ensure infectivity. Perturbating the membrane of viruses using exogenous lipids can be envisioned as a generic way to reduce their infectivity. In this context, the potential of exogenous lipids to be used against enveloped virus infectivity would be indicated by the resulting physical stress imposed to the viral membrane, and conical lipids, i.e. lyso-lipids, would be expected to generate stronger biophysical disturbances. We confirm that when treated with lyso-lipids the infectivity three strains of influenza virus (avian H2N3, equine H3N8 or pandemic human influenza H1N1) is reduced by up to 99% in a cell-based model. By contrast, lipids with a similar head group but two aliphatic chains were less effective (reducing infection by only 40–50%). This work opens a new path to merge concepts from different research fields, i.e. ‘soft matter physics' and virology

Longitudinal study of Asian elephants, Elephas maximus, indicates intermittent shedding of elephant endotheliotropic herpesvirus 1 during pregnancy

Introduction: EEHV-1 is a viral infection of elephants that has been associated with a fatal haemorrhagic syndrome in Asian elephants. Previous studies have suggested that pregnant animals may shed more virus than non-pregnant animals. Methods: This study examined whether pregnancy affected the frequency or magnitude of shedding of elephant endotheliotropic herpesvirus 1 (EEHV1) using Taq man real-time PCR on trunk washes from four female elephants from a UK collection over three time periods between 2011 and 2014. These periods included pregnancies in two animals (period 1 and period 3). Behavioural observations made by keepers were also assessed. Results: During period 1 there was a high degree of social hierarchical instability which led to a hierarchy change, and was associated with aggressive behaviour. Also during period 1 EEHV-1 shedding was of a higher magnitude and frequency than in the latter two time periods. Conclusions: These results suggest that there is no clear relationship between shedding and pregnancy, and that behavioural stressors may be related to an increase in EEHV-1 shedding

Comparative distribution of human and avian type sialic acid influenza receptors in the pig

<p>Abstract</p> <p>Background</p> <p>A major determinant of influenza infection is the presence of virus receptors on susceptible host cells to which the viral haemagglutinin is able to bind. Avian viruses preferentially bind to sialic acid α2,3-galactose (SAα2,3-Gal) linked receptors, whereas human strains bind to sialic acid α2,6-galactose (SAα2,6-Gal) linked receptors. To date, there has been no detailed account published on the distribution of SA receptors in the pig, a model host that is susceptible to avian and human influenza subtypes, thus with potential for virus reassortment. We examined the relative expression and spatial distribution of SAα2,3-GalG(1-3)GalNAc and SAα2,6-Gal receptors in the major organs from normal post-weaned pigs by binding with lectins <it>Maackia amurensis agglutinins </it>(MAA II) and <it>Sambucus nigra agglutinin </it>(SNA) respectively.</p> <p>Results</p> <p>Both SAα2,3-Gal and SAα2,6-Gal receptors were extensively detected in the major porcine organs examined (trachea, lung, liver, kidney, spleen, heart, skeletal muscle, cerebrum, small intestine and colon). Furthermore, distribution of both SA receptors in the pig respiratory tract closely resembled the published data of the human tract. Similar expression patterns of SA receptors between pig and human in other major organs were found, with exception of the intestinal tract. Unlike the limited reports on the scarcity of influenza receptors in human intestines, we found increasing presence of SAα2,3-Gal and SAα2,6-Gal receptors from duodenum to colon in the pig.</p> <p>Conclusions</p> <p>The extensive presence of SAα2,3-Gal and SAα2,6-Gal receptors in the major organs examined suggests that each major organ may be permissive to influenza virus entry or infection. The high similarity of SA expression patterns between pig and human, in particular in the respiratory tract, suggests that pigs are not more likely to be potential hosts for virus reassortment than humans. Our finding of relative abundance of SA receptors in the pig intestines highlights a need for clarification on the presence of SA receptors in the human intestinal tract.</p

Development of Virus-like Particle Plant-Based Vaccines against Avian H5 and H9 Influenza A Viruses

Avian influenza A virus (AIV) is a significant cause of mortality in poultry, causing substantial economic loss, particularly in developing countries, and has zoonotic potential. For example, highly pathogenic avian influenza (HPAI) viruses of the H5 subtype have been circulating in Egypt for around two decades. In the last decade, H5N1 viruses of clade 2.2.1 have been succeeded by the antigenically distinct H5N8 clade 2.3.4.4b viruses. Furthermore, H9N2 viruses co-circulate with the H5N8 viruses in Egyptian poultry. It is widely recognised that effective vaccination against IAV requires a close antigenic match between the vaccine and viruses circulating in the field. Therefore, approaches to develop cost-effective vaccines that can be rapidly adapted to local virus strains are required for developing countries such as Egypt. In this project, the haemagglutinin (HA) proteins of Egyptian H5 and H9 viruses were expressed by transient transfection of plants (Nicotiana benthamiana). The formation of virus-like particles (VLPs) was confirmed by transmission electron microscopy. Mice were immunised with four doses of either H5 or H9 VLPs with adjuvant. Antibody and cellular immune responses were measured against the corresponding recombinant protein using ELISA and enzyme-linked immunosorbent assay (ELISpot), respectively. Chickens were immunised with one dose of H5 VLPs, eliciting HA-specific antibodies measured by ELISA and a pseudotyped virus neutralisation test using a heterologous H5 HA. In conclusion, plant-based VLP vaccines have potential for producing an effective vaccine candidate within a short time at a relatively low cost

An Analysis of the Environments of FU Orionis Objects with Herschel

We present Herschel-HIFI, SPIRE, and PACS 50-670 {\mu}m imaging and spectroscopy of six FU Orionis-type objects and candidates (FU Orionis, V1735 Cyg, V1515 Cyg, V1057 Cyg, V1331 Cyg, and HBC 722), ranging in outburst date from 1936-2010, from the "FOOSH" (FU Orionis Objects Surveyed with Herschel) program, as well as ancillary results from Spitzer-IRS and the Caltech Submillimeter Observatory. In their system properties (Lbol, Tbol, line emission), we find that FUors are in a variety of evolutionary states. Additionally, some FUors have features of both Class I and II sources: warm continuum consistent with Class II sources, but rotational line emission typical of Class I, far higher than Class II sources of similar mass/luminosity. Combining several classification techniques, we find an evolutionary sequence consistent with previous mid-IR indicators. We detect [O I] in every source at luminosities consistent with Class 0/I protostars, much greater than in Class II disks. We detect transitions of 13CO (J_up of 5 to 8) around two sources (V1735 Cyg and HBC 722) but attribute them to nearby protostars. Of the remaining sources, three (FU Ori, V1515 Cyg, and V1331 Cyg) exhibit only low-lying CO, but one (V1057 Cyg) shows CO up to J = 23 - 22 and evidence for H2O and OH emission, at strengths typical of protostars rather than T Tauri stars. Rotational temperatures for "cool" CO components range from 20-81 K, for ~ 10^50 total CO molecules. We detect [C I] and [N II] primarily as diffuse emission.Comment: 31 pages, 15 figures; accepted to Ap

Rapid death of duck cells infected with influenza: a potential mechanism for host resistance to H5N1

Aquatic birds are the natural reservoir for most subtypes of influenza A, and a source of novel viruses with the potential to cause human pandemics, fatal zoonotic disease or devastating epizootics in poultry. It is well recognised that waterfowl typically show few clinical signs following influenza A infection, in contrast, terrestrial poultry such as chickens may develop severe disease with rapid death following infection with highly pathogenic avian influenza. This study examined the cellular response to influenza infection in primary cells derived from resistant (duck) and susceptible (chicken) avian hosts. Paradoxically, we observed that duck cells underwent rapid cell death following infection with low pathogenic avian H2N3, classical swine H1N1 and ‘classical' highly pathogenic H5N1 viruses. Dying cells showed morphological features of apoptosis, increased DNA fragmentation and activation of caspase 3/7. Following infection of chicken cells, cell death occurred less rapidly, accompanied by reduced DNA fragmentation and caspase activation. Duck cells produced similar levels of viral RNA but less infectious virus, in comparison with chicken cells. Such rapid cell death was not observed in duck cells infected with a contemporary Eurasian lineage H5N1 fatal to ducks. The induction of rapid death in duck cells may be part of a mechanism of host resistance to influenza A, with the loss of this response leading to increased susceptibility to emergent strains of H5N1. These studies provide novel insights that should help resolve the long-standing enigma of host–pathogen relationships for highly pathogenic and zoonotic avian influenza