Biology subjects

Skinner, M. A.

Publications and source records attributed to Skinner, M. A..

9 recordsLinked to original sources

bioRxiv2024

Targeting DNA-PK is a highly conserved poxvirus innate immune evasion mechanism

The sensing of viral nucleic acid by pattern recognition receptors (PRRs) is essential for initiation of a type-I interferon response against infection. Intracellular DNA sensing PRRs are responsible for initiating innate immune responses to poxviruses and other double-stranded DNA viruses. Poxviruses, in turn, encode an armoury of immunomodulators that inhibit this host defence mechanism. DNA-dependent protein kinase (DNA-PK) is an essential component of the cGAS/STING-dependent viral DNA sensing machinery that leads to the initiation of a type-I interferon response during poxvirus infection. Poxviruses counter this host sensing mechanism using the C4/C10 family of proteins that target DNA-PK, interfering with its ability to bind viral DNA. Although the DNA-PK complex, known also for its role in double strand break repair, is conserved across multiple taxa, its function in innate immunity outside mammals is unexplored. Here we analysed the contribution of DNA-PK to poxvirus DNA sensing in chickens, a species that is evolutionarily distant from mammals, but that is also infected by poxviruses. We found that DNA-PK functions as a DNA sensor in chickens, and this process is countered by C4/C10 family members found in fowlpox virus. This host/pathogen interaction is conserved across a broader range of species than other mechanisms of poxvirus antagonism of innate immune sensing, which may reflect the difficulty of the host in evolving escape mechanisms that interfere with a protein that is essential for maintenance of genomic stability.

immunology↗

bioRxiv2024

PluMu - a Mu-like bacteriophage infecting Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae is the causative agent of pleuropneumonia, an economically important lung disease of pigs. In draft genomes of two Cypriot clinical A. pleuropneumoniae isolates (MIDG3457 and MIDG3459), we previously identified single genomic regions with homology to Mu-like bacteriophage and presented preliminary evidence of active phage. Here, updated Phastest genomic analysis identified two loci in both MIDG3457 and MIDG3459 that were predicted to encode proteins with high homology to, and whose organization was characteristic of, Mu-like phages. Phylogenetically, the closest matches were with Mannheimia Vb and Glaesserella SuMu phages. Phastest scored the loci as "complete", indicating they produced active phage. PCR amplification of the Mu-like phage c and tail genes from DNase-treated polyethylene glycol 8000 (PEG)-precipitated supernatants of MIDG3457 and MIDG3459 (grown in either Brain Heart Infusion-NAD or Graces Insect Medium-NAD broth) indicated the presence of intact virions. The phages from MIDG3457 and MIDG3459 were named PluMu 3457-1, 3457-2, and PluMu 3459-1 and PluMu 3459-2, respectively. Transmission electron microscopy (TEM) of PEG-precipitated supernatants of broth-grown MIDG3459 identified virions with icosahedral heads and tails, consistent with other Mu-like phages. We conclude that MIDG3459 produces an active Mu- like phage.

microbiology↗

bioRxiv2021

Transcriptomic analysis reveals that severity of infectious bursal disease in White Leghorn inbred chicken lines is associated with greater bursal inflammation in vivo and more rapid induction of pro-inflammatory responses in primary bursal cells stimulated ex vivo.

In order to better understand differences in the outcome of infectious bursal disease virus (IBDV) infection, we inoculated a very virulent (vv) strain into White Leghorn chickens of inbred line W that was previously reported to experience over 24% flock mortality, and three inbred lines (15I, C.B4 and 0) that were previously reported to display no mortality. Within each experimental group, some individuals experienced more severe disease than others but line 15I birds experienced milder disease based on average clinical scores, percentage of birds with gross pathology, average bursal lesion scores and average peak bursal virus titre. RNA-Seq analysis revealed that more severe disease in line W was associated with significant up-regulation of pathways involved in inflammation, cytoskeletal regulation by Rho GTPases, nicotinic acetylcholine receptor signaling, and Wnt signaling in the bursa compared to line 15I. Primary bursal cell populations isolated from uninfected line W birds contained a significantly greater percentage of KUL01+ macrophages than cells isolated from line 15I birds (p<0.01) and, when stimulated ex vivo with LPS, showed more rapid up-regulation of pro-inflammatory gene expression than those from line 15I birds. We hypothesize that a more rapid induction of pro-inflammatory cytokine responses in bursal cells following IBDV infection leads to more severe disease in line W birds than in line 15I.

microbiology↗

bioRxiv2020

Modulation of early host innate immune response by a Fowlpox virus (FWPV) lateral body protein

The avian pathogen, fowlpox virus (FWPV) has been successfully used as vaccine vector in poultry and humans but relatively little is known about its ability to modulate host antiviral immune responses in these hosts, which are replication permissive and non-permissive, respectively. FWPV is highly resistant to avian type I interferon (IFN) and able to completely block the host IFN-response. Microarray screening of host IFN-regulated gene expression in cells infected with 59 different, non-essential FWPV gene knock-out mutants revealed that FPV184 confers immunomodulatory capacity. We report that FPV184-knockout virus (FWPV{Delta}184) induces the cellular IFN response as early as 2 hours post-infection. The wild-type, uninduced phenotype can be rescued by transient expression of FPV184 in FWPV{Delta}184-infected cells. Ectopic expression of FPV184 inhibited polyI:C activation of the chicken IFN-{beta} promoter and IFN- activation of the chicken Mx promoter. Confocal and correlative super-resolution light and electron microscopy demonstrated that FPV184 has a functional nuclear localisation signal domain and is packaged in the lateral bodies of the virions. Taken together, these results provide a paradigm for a late poxvirus structural protein packaged in the lateral bodies and capable of supressing IFN induction early during the next round of infection.

microbiology↗

bioRxiv2020

Chicken cGAS senses fowlpox virus infection and regulates macrophage effector function

The anti-viral immune response is dependent on the ability of infected cells to sense foreign nucleic acids. In multiple species, the pattern recognition receptor (PRR) cyclic GMP-AMP synthase (cGAS) senses viral DNA as an essential component of the innate response. cGAS initiates a range of signalling outputs that are dependent on generation of the second messenger cGAMP that binds to the adaptor protein stimulator of interferon genes (STING). Here we show that in chicken macrophages, the cGAS/STING pathway is essential not only for the production of type-I interferons in response to intracellular DNA stimulation, but also for regulation of macrophage effector functions including the expression of MHC-II and co-stimulatory molecules. In the context of fowlpox, an avian DNA virus infection, the cGAS/STING pathway was found to be responsible for type-I interferon production and MHC-II transcription. The sensing of fowlpox virus DNA is therefore essential for mounting an anti-viral response in chicken cells and for regulation of a specific set of macrophage effector functions.

immunology↗

bioRxiv2019

The stronger downregulation of in vitro and in vivo innate antiviral responses by a very virulent strain of infectious bursal disease virus (IBDV), compared to a classical strain, is mediated, in part, by the VP4 protein.

IBDV is economically important to the poultry industry. Very virulent (vv) strains cause higher mortality rates than other strains for reasons that remain poorly understood. In order to provide more information on IBDV disease outcome, groups of chickens (n=18) were inoculated with the vv strain, UK661, or the classical strain, F52/70. Birds infected with UK661 had a lower survival rate (50%) compared to F52/70 (80%). There was no difference in peak viral replication in the bursa of Fabricius (BF), but the expression of chicken IFN{beta}, MX1 and IL-8 was significantly lower in the BF of birds infected with UK661 compared to F52/70 (p<0.05) as quantified by RTqPCR, and this trend was also observed in DT40 cells infected with UK661 or F52/70 (p<0.05). The induction of expression of type I IFN in DF-1 cells stimulated with polyI:C (measured by an IFN-{beta} luciferase reporter assay) was significantly reduced in cells expressing ectopic VP4 from UK661 (p<0.05), but was higher in cells expressing ectopic VP4 from F52/70. Cells infected with a chimeric recombinant IBDV carrying the UK661-VP4 gene in the background of PBG98, an attenuated vaccine strain that induces high levels of innate responses (PBG98-VP4UK661) also showed a reduced level of IFN and IL-8 compared to cells infected with a chimeric virus carrying the F52/70-VP4 gene (PBG98-VP4F52/70), and birds infected with PBG98-VP4UK661 also had a reduced expression of IFN in the BF compared to birds infected with PBG98-VP4F52/70. Taken together, these data demonstrate that UK661 induced the expression of lower levels of anti-viral type I IFN and proinflammatory genes than the classical strain in vitro and in vivo and this was, in part, due to strain-dependent differences in the VP4 protein.

microbiology↗

bioRxiv2019

The MHC class-II HLA-DR receptor mediates bat influenza A-like H17N10 virus entry into mammalian cells

Bats are notorious reservoirs of diverse, potentially zoonotic viruses, exemplified by the evolutionarily distinct, influenza A-like viruses H17N10 and H18N11 (BatIVs). The surface glycoproteins [haemagglutinin (H) and neuraminidase (N)] of BatIVs neither bind nor cleave sialic acid receptors, which suggests that these viruses employ cell attachment and entry mechanisms that differ from those of classical influenza A viruses (IAVs). Identifying the cellular factors that mediate entry and determine susceptibility to infection will help assess the host range of BatIVs. Here, we investigated a range of cell lines from different species for their susceptibility to infection by pseudotyped viruses (PV) bearing bat H17 and/or N10 envelope glycoproteins. We show that a number of human haematopoietic cancer cell lines and the canine kidney MDCK II (but not MDCK I) cells are susceptible to H17-pseudotypes (H17-PV). We observed with microarrays and qRT-PCR that the dog leukocyte antigen DLA-DRA mRNA is over expressed in late passaged parental MDCK and commercial MDCK II cells, compared to early passaged parental MDCK and MDCK I cells, respectively. The human orthologue HLA-DRA encodes the alpha subunit of the MHC class II HLA-DR antigen-binding heterodimer. Small interfering RNA- or neutralizing antibody-targeting HLA-DRA, drastically reduced the susceptibility of Raji B cells to H17-PV. Conversely, over expression of HLA-DRA and its paralogue HLA-DRB1 on the surface of the unsusceptible HEK293T/17 cells conferred susceptibility to H17-PV. The identification of HLA-DR as an H17N10 entry mediator will contribute to a better understanding of the tropism of the virus and will elucidate its zoonotic transmission.

microbiology↗

bioRxiv2019

Avian ANP32B does not support influenza A virus polymerase and influenza A virus relies exclusively on ANP32A in chicken cells.

Influenza A viruses (IAV) are subject to species barriers that prevent frequent zoonotic transmission and pandemics. One of these barriers is the poor activity of avian IAV polymerases in human cells. Differences between avian and mammalian ANP32 proteins underlie this host range barrier. Human ANP32A and ANP32B homologues both support function of human-adapted influenza polymerase but do not support efficient activity of avian IAV polymerase which requires avian ANP32A. We show here that avian ANP32B is evolutionarily distinct from mammalian ANP32B, and that chicken ANP32B does not support IAV polymerase activity even of human-adapted viruses. Consequently, IAV does not replicate in chicken cells that lack ANP32A. Amino acid differences in LRR5 domain accounted for the inactivity of chicken ANP32B. Transfer of these residues to chicken ANP32A abolished support of IAV polymerase. Understanding ANP32 function will help develop antiviral strategies and aid the design of influenza virus resistant genome edited chickens.

microbiology↗

bioRxiv2017

Differential gene expression in chicken primary B cells infected ex vivo with attenuated and very virulent strains of infectious bursal disease virus (IBDV)

Infectious bursal disease virus (IBDV) belongs to the family Birnaviridae and is economically important to the poultry industry worldwide. IBDV infects B cells in the bursa of Fabricius (BF), causing immunosuppression and morbidity in young chickens. In addition to strains that cause classical Gumboro disease, the so-called very virulent (vv) strain, also in circulation, causes more severe disease and increased mortality. IBDV has traditionally been controlled through the use of live attenuated vaccines, with attenuation resulting from serial passage in non-lymphoid cells. However, the factors that contribute to the vv or attenuated phenotypes are poorly understood. In order to address this, we aimed to investigate host cell-IBDV interactions using a recently described chicken primary B cell model, where chicken B cells are harvested from the BF and cultured ex vivo in the presence of chicken CD40L. We demonstrated that these cells could support the replication of IBDV when infected ex vivo in the laboratory. Furthermore, we evaluated the gene expression profiles of B cells infected with an attenuated strain (D78) and a very virulent strain (UK661) by microarray. We found that key genes involved in B cell activation and signaling (TNFSF13B, CD72 and GRAP) were down-regulated following infection relative to mock, which we speculate could contribute to IBDV-mediated immunosuppression. Moreover, cells responded to infection by expressing antiviral type I IFNs and IFN-stimulated genes, but the induction was far less pronounced upon infection with UK661, which we speculate could contribute to its virulence.

microbiology↗