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SARS-CoV-2 attacks various organs, most destructively the lung, and cellular entry requires two host cell surface proteins: ACE2 and TMPRSS2. Downregulation of one or both of these is thus a potential therapeutic approach for COVID-19. TMPRSS2 is a known target of the androgen receptor, a ligand-activated transcription factor; androgen receptor activation increases TMPRSS2 levels in various tissues, most notably prostate. We show here that treatment with the antiandrogen enzalutamide—a well-tolerated drug widely used in advanced prostate cancer—reduces TMPRSS2 levels in human lung cells and in mouse lung. Importantly, antiandrogens significantly reduced SARS-CoV-2 entry and infection in lung cells. In support of this experimental data, analysis of existing datasets shows striking co-expression of AR and TMPRSS2, including in specific lung cell types targeted by SARS-CoV-2. Together, the data presented provides strong evidence to support clinical trials to assess the efficacy of antiandrogens as a treatment option for COVID-19., TMPRSS2 is regulated by androgen receptor signalling in the prostate, however it is unclear if blocking this signalling is beneficial in the context of SARS-CoV-2 lung infection. Here the authors show that antiandrogen treatment downregulates TMPRSS2 in the lung and reduces viral entry and infection.

Involvement of macrophages in the SARS-CoV-2-associated cytokine storm, the excessive secretion of inflammatory/anti-viral factors leading to the acute respiratory distress syndrome (ARDS) in COVID-19 patients, is unclear. In this study, we sought to characterize the interplay between the virus and primary human monocyte-derived macrophages (MDM). MDM were stimulated with recombinant IFN-α and/or infected with either live or UV-inactivated SARS-CoV-2 or with two reassortant influenza viruses containing external genes from the H1N1 PR8 strain and heterologous internal genes from a highly pathogenic avian H5N1 or a low pathogenic human seasonal H1N1 strain. Virus replication was monitored by qRT-PCR for the E viral gene for SARS-CoV-2 or M gene for influenza and TCID50 or plaque assay, and cytokine levels were assessed semiquantitatively with qRT-PCR and a proteome cytokine array. We report that MDM are not susceptible to SARS-CoV-2 whereas both influenza viruses replicated in MDM, albeit abortively. We observed a modest cytokine response in SARS-CoV-2 infected MDM with notable absence of IFN-β induction, which was instead strongly induced by the influenza viruses. Pre-treatment of MDM with IFN-α enhanced proinflammatory cytokine expression upon infection. Together, the findings concur that the hyperinflammation observed in SARS-CoV-2 infection is not driven by macrophages.

Haemagglutinin and neuraminidase surface glycoproteins of the bat influenza H17N10 virus neither bind to nor cleave sialic acid receptors, indicating that this virus employs cell entry mechanisms distinct from those of classical influenza A viruses. We observed that certain human haematopoietic cancer cell lines and canine MDCK II cells are susceptible to H17-pseudotyped viruses. We identified the human HLA-DR receptor as an entry mediator for H17 pseudotypes, suggesting that H17N10 possesses zoonotic potential.

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 &lt, 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.

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 (pex 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.

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.

The COVID-19 pandemic, caused by the novel human coronavirus SARS-CoV-2 coronavirus, attacks various organs but most destructively the lung. It has been shown that SARS-CoV-2 entry into lung cells requires two host cell surface proteins: ACE2 and TMPRSS2. Downregulation of one or both of these is thus a potential therapeutic approach for COVID-19. TMPRSS2 is a known target of the androgen receptor, a ligand-activated transcription factor; activation of the androgen receptor increases TMPRSS2 levels in various tissues, most notably the prostate. We show here that treatment with the antiandrogen enzalutamide – a well-tolerated drug widely used in advanced prostate cancer – reduces TMPRSS2 levels in human lung cells. Further, enzalutamide treatment of mice dramatically decreased Tmprss2 levels in the lung. To determine therapeutic potential, we assessed uptake of SARS-CoV-2 Spike protein pseudotyped lentivirus and live SARS-CoV-2 into human lung cells and saw a significant reduction in viral entry and infection upon treatment with the antiandrogens enzalutamide and bicalutamide. In support of this new experimental data, analysis of existing datasets shows striking co-expression of AR and TMPRSS2, including in specific lung cell types that are targeted by SARS-CoV-2. Together, the data presented provides strong evidence to support clinical trials to assess the efficacy of antiandrogens as a treatment option for COVID-19.

The avian pathogen fowlpox virus (FWPV) has been successfully used as a 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 nonpermissive, 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, nonessential FWPV gene knockout mutants revealed that FPV184 confers immunomodulatory capacity. We report that the FPV184-knockout virus (FWPV&Delta, 184) induces the cellular IFN response as early as 2 h postinfection. 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-&alpha, activation of the chicken Mx1 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, capable of suppressing IFN induction early during the next round of infection.

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 (FWPVΔ184) 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 FWPVΔ184-infected cells. Ectopic expression of FPV184 inhibited polyI:C activation of the chicken IFN-β promoter and IFN-α 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.

Hepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma (HCC) worldwide. The prolyl hydroxylase domain (PHD)-hypoxia inducible factor (HIF) pathway is a key mammalian oxygen sensing pathway and is frequently perturbed by pathological states including infection and inflammation. We discovered a significant upregulation of hypoxia regulated gene transcripts in patients with chronic hepatitis B (CHB) in the absence of liver cirrhosis. We used state-of-the-art in vitro and in vivo HBV infection models to evaluate a role for HBV infection and the viral regulatory protein HBx to drive HIF-signalling. HBx had no significant impact on HIF expression or associated transcriptional activity under normoxic or hypoxic conditions. Furthermore, we found no evidence of hypoxia gene expression in HBV de novo infection, HBV infected human liver chimeric mice or transgenic mice with integrated HBV genome. Collectively, our data show clear evidence of hypoxia gene induction in CHB that is not recapitulated in existing models for acute HBV infection, suggesting a role for inflammatory mediators in promoting hypoxia gene expression.

The diversity of subtypes within Influenza A recently expanded with identification of H17N10 and H18N11 from bats. To study the tropism and zoonotic potential of these viruses, we successfully produced lentiviral pseudotypes bearing haemagglutinin H17 and neuraminidase N10. We investigated a range of cell lines from different species for their susceptibility to infection by these pseudotypes and show that a number of human haematopoietic cancer cell lines and the canine kidney MDCK II (but not MDCK I) cells are susceptible. Using microarrays and qRT-PCR we show 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 unsusceptible HEK293T/17 cells conferred susceptibility to H17-PV. The identification of HLA-DR as an H17N10 entry mediator will contribute to understanding the tropism of the virus and help to elucidate its zoonotic transmission. We also show that H17 pseudotypes can be efficiently neutralised by the broadly-neutralizing HA2 stalk monoclonal antibodies CR9114 and FI6. The lentiviral pseudotype system is a useful research tool, amenable for investigation of bat influenza tropism, restriction and pandemic preparedness, without safety issues of producing a replication-competent virus, to which the human population is naïve.

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 IFNβ, MX1 and IL-8 was significantly lower in the BF of birds infected with UK661 compared to F52/70 (pUK661) also showed a reduced level of IFNα 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-VP4UK661also had a reduced expression of IFNα 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 strainin vitroandin vivoand this was, in part, due to strain-dependent differences in 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 IFNα, IFNβ, 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-β 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 IFNα and IL-8 compared to cells infected with a chimeric virus carrying the F52/70-VP4 gene (PBG98-VP4F52/70) (p < 0.01), and birds infected with PBG98-VP4UK661 also had a reduced expression of IFNα in the BF compared to birds infected with PBG98-VP4F52/70 (p < 0.05). 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.

Chicken anaemia virus (CAV) is a lymphotropic virus that causes anaemia and immunosuppression in chickens. Previously, we proposed that CAV evades host antiviral responses in vivo by disrupting T-cell signalling, but the precise cellular targets and modes of action remain elusive. In this study, we examined gene expression in Marek's disease virus-transformed chicken T-cell line MSB-1 after infection with CAV using both a custom 5K immune-focused microarray and quantitative real-time PCR at 24, 48 and 72 h post-infection. The data demonstrate an intricate equilibrium between CAV and the host gene expression, displaying subtle but significant modulation of transcripts involved in the T-cell, inflammation and NF-κB signalling cascades. CAV efficiently blocked the induction of type-I interferons and interferon-stimulated genes at 72 h. The cell expression pattern implies that CAV subverts host antiviral responses and that the transformed environment of MSB-1 cells offers an opportunistic advantage for virus growth.

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.

Haemagglutinin and neuraminidase surface glycoproteins of the bat influenza H17N10 virus neither bind to nor cleave sialic acid receptors, indicating that this virus employs cell entry mechanisms distinct from those of classical influenza A viruses. We observed that certain human haematopoietic cancer cell lines and canine MDCK II cells are susceptible to H17-pseudotyped viruses. We identified the human HLA-DR receptor as an entry mediator for H17 pseudotypes, suggesting that H17N10 possesses zoonotic potential.

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.

The discovery of mammalian pluripotent embryonic stem cells (ESC) has revolutionised cell research and regenerative medicine. More recently discovered chicken ESC (cESC), though less intensively studied, are increasingly popular as vaccine substrates due to a dearth of avian cell lines. Information on the comparative performance of cESC with common vaccine viruses is limited. Using RNA-sequencing, we compared cESC transcriptional programmes elicited by stimulation with chicken type I interferon or infection with vaccine viruses routinely propagated in primary chicken embryo fibroblasts (CEF). We used poxviruses (fowlpox virus (FWPV) FP9, canarypox virus (CNPV), and modified vaccinia virus Ankara (MVA)) and a birnavirus (infectious bursal disease virus (IBDV) PBG98). Interferon-stimulated genes (ISGs) were induced in cESC to levels comparable to those in CEF and immortalised chicken fibroblast DF-1 cells. cESC are permissive (with distinct host transcriptional responses) to MVA, FP9, and CNPV but, surprisingly, not to PBG98. MVA, CNPV, and FP9 suppressed innate immune responses, while PBG98 induced a subset of ISGs. Dysregulation of signalling pathways (i.e., NF&kappa, B, TRAF) was observed, which might affect immune responses and viral replication. In conclusion, we show that cESC are an attractive alternative substrate to study and propagate poxvirus recombinant vaccine vectors.

The diversity of subtypes within the Influenza A virus genus has recently expanded with the identification of H17N10 and H18N11 from bats. In order to further study the tropism and zoonotic potential of these viruses, we have successfully produced lentiviral pseudotypes bearing both H17 and N10. These pseudotypes were shown to be efficiently neutralized by the broadly-neutralizing monoclonal antibodies CR9114 and FI6. Our studies also confirm previous reports that H17 does not use sialic acid as its cellular receptor, as pseudotypes bearing the H17 envelope glycoprotein are released into the cell supernatant in the absence of neuraminidase. However, we demonstrate that N10 facilitates heterosubtypic (H5 and H7) influenza hemagglutinin-bearing pseudotype release in the absence of another source of neuraminidase, significantly increasing luciferase pseudotype production titres. Despite this, N10 shows no activity in the enzyme-linked lectin assay used for traditional sialidases. These findings suggest that this protein plays an important role in viral egress, but is perhaps involved in further accessory roles in the bat influenza lifecycle that are yet to be discovered. Thus we show the lentiviral pseudotype system is a useful research tool, and amenable for investigation of bat influenza tropism, restriction and sero-epidemiology, without the constraints or safety issues with producing a replication-competent virus, to which the human population is naive.

Fowlpox virus is the type species of an extensive and poorly-defined group of viruses isolated from more than 200 species of birds, together comprising the avipoxvirus genus of the poxvirus family. Long known as a significant poultry pathogen, vaccines developed in the early and middle years of the twentieth century led to its effective eradication as a problem to commercial production in temperate climes in developed western countries (such that vaccination there is now far less common). Transmitted mechanically by biting insects, it remains problematic, causing significant losses to all forms of production (from backyard, through extensive to intensive commercial flocks), in tropical climes where control of biting insects is difficult. In these regions, vaccination (via intradermal or subcutaneous, and increasingly in ovo, routes) remains necessary. Although there is no evidence that more than a single serotype exists, there are poorly-described reports of outbreaks in vaccinated flocks. Whether this is due to inadequate vaccination or penetrance of novel variants remains unclear. Some such outbreaks have been associated with strains carrying endogenous, infectious proviral copies of the retrovirus reticuloendotheliosis virus (REV), which might represent a pathotypic (if not newly emerging) variant in the field. Until more is known about the phylogenetic structure of the avipoxvirus genus (by more widespread genome sequencing of isolates from different species of birds) it remains difficult to ascertain the risk of novel avipoxviruses emerging from wild birds (and/or by recombination/mutation) to infect farmed poultry.

International audience; Influenza pandemics occur unpredictably when zoonotic influenza viruses with novel antigenicity acquire the ability to transmit amongst humans. Host range breaches are limited by incompatibilities between avian virus components and the human host. Barriers include receptor preference, virion stability and poor activity of the avian virus RNA-dependent RNA polymerase in human cells. Mutants of the heterotrimeric viral polymerase components, particularly PB2 protein, are selected during mammalian adaptation, but their mode of action is unknown. We show that a species-specific difference in host protein ANP32A accounts for the suboptimal function of avian virus polymerase in mammalian cells. Avian ANP32A possesses an additional 33 amino acids between the leucine-rich repeats and carboxy-terminal low-complexity acidic region domains. In mammalian cells, avian ANP32A rescued the suboptimal function of avian virus polymerase to levels similar to mammalian-adapted polymerase. Deletion of the avian-specific sequence from chicken ANP32A abrogated this activity, whereas its insertion into human ANP32A, or closely related ANP32B, supported avian virus polymerase function. Substitutions, such as PB2(E627K), were rapidly selected upon infection of humans with avian H5N1 or H7N9 influenza viruses, adapting the viral polymerase for the shorter mammalian ANP32A. Thus ANP32A represents an essential host partner co-opted to support influenza virus replication and is a candidate host target for novel antivirals.

Hepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma (HCC) worldwide. The prolyl hydroxylase domain (PHD)-hypoxia inducible factor (HIF) pathway is a key mammalian oxygen sensing pathway and is frequently perturbed by pathological states including infection and inflammation. We discovered a significant upregulation of hypoxia regulated gene transcripts in patients with chronic hepatitis B (CHB) in the absence of liver cirrhosis. We used state-of-the-art in vitro and in vivo HBV infection models to evaluate a role for HBV infection and the viral regulatory protein HBx to drive HIF-signalling. HBx had no significant impact on HIF expression or associated transcriptional activity under normoxic or hypoxic conditions. Furthermore, we found no evidence of hypoxia gene expression in HBV de novo infection, HBV infected human liver chimeric mice or transgenic mice with integrated HBV genome. Collectively, our data show clear evidence of hypoxia gene induction in CHB that is not recapitulated in existing models for acute HBV infection, suggesting a role for inflammatory mediators in promoting hypoxia gene expression.

Evidence of an association between added sugars (AS) and the risk of obesity has triggered public health bodies to develop strategies enabling consumers to manage their AS intake. The World Health Organisation (WHO) has strongly recommended a reduction of free sugars to 10% of total dietary energy (TE) and conditionally recommended a reduction to 5% TE to achieve health benefits. Despite food labelling being a policy tool of choice in many countries, there is no consensus on the mandatory addition of AS to the nutrition panel of food labels. An online survey was conducted to explore consumer ability to identify AS on food labels and to investigate consumer awareness of the WHO guidelines in relation to sugar intakes. The questionnaire was tested for participant comprehension using face-to-face interviews prior to conducting the online study. The online survey was conducted in Northern Ireland during May 2015 and was completed by a convenient sample of 445 subjects. Results showed that just 4% of respondents correctly classified 10 or more ingredients from a presented list of 13 items, while 65% of participants were unaware of the WHO guidelines for sugar intake. It may be timely to reopen dialogue on inclusion of AS on food product nutrition panels.

Preventing microbial contamination during dehiding is challenging, and skinning methods are of critical importance for the hygienic status of beef carcasses. Two skinning methods are usually employed: upward hide pulling (UHP) and downward hide pulling (DHP). This study has compared the microbiological contamination of carcasses using both systems in a beef processing plant in the process of changing its dehiding method from UHP to DHP. 100 cm(2) areas from eight carcass sites (ham, chuck, rump, bung, flank, brisket, shin and neck) were sampled on 36 skinned carcasses dehided by each technique. Total viable counts (TVCs) and Enterobacteriaceae counts for each site were determined. No significant differences were observed in total (pooled-samples) carcass contamination regardless of the method used. However, significant differences (p

Alkali stress is an important means of inactivating undesirable pathogens in a wide range of situations. Unfortunately, Listeria monocytogenes can launch an alkaline tolerance response, significantly increasing persistence of the pathogen in such environments. This study compared transcriptome patterns of alkali and non-alkali-stressed L. monocytogenes 10403S cells, to elucidate the mechanisms by which Listeria adapts and/or grows during short- or long-term alkali stress. Transcription profiles associated with alkali shock (AS) were obtained by DNA microarray analysis of midexponential cells suspended in pH 9 media for 15, 30, or 60 min. Transcription profiles associated with alkali adaptation (AA) were obtained similarly from cells grown to midexponential phase at pH 9. Comparison of AS and AA transcription profiles with control cell profiles identified a high number of differentially regulated open-reading frames in all tested conditions. Rapid (15 min) changes in expression included upregulation of genes encoding for multiple metabolic pathways (including those associated with Na+/H+ antiporters), ATP-binding cassette transporters of functional compatible solutes, motility, and virulence-associated genes as well as the σ(B) controlled stress resistance network. Slower (30 min and more) responses to AS and adaptation during growth in alkaline conditions (AA) involved a different pattern of changes in mRNA concentrations, and genes involved in proton export.

The changes in cell surface properties associated with alkali stress can significantly disrupt cell metabolism and structures, preventing effective interactions between bacterial cells and their environment. Listeria monocytogenes is known to display an adaptive response to alkali stress that enhances its capacity to more effectively survive subsequent severe alkali challenge. In this study, we examined the effects of adaptation to alkali conditions (pH 9.5/ 1h) in reducing detrimental effects in hydrophobicity and cell morphology in Listeria monocytogenes 10403S during severe (subsequent) short-term alkali challenge (pH 12.0/ 1h). Severe alkali challenge induced larger reductions in hydrophobicity (i.e., lower MATH values) in non adapted control cells than in mild alkali adapted cells. SEM revealed greater morphological diver- sity in suspensions of non alkali adapted cells than in suspensions of mild alkali adapted cells, i.e., a larger proportion of alkali adapted cells retained the normal (bacillary) morphology. Non adapted cells displayed considerable morphological heterogeneity including elongation, rupture and cellular deformation. Short-term alkaline adaptation in L. monocytogenes involves direct changes in the cell surface properties and organisation which facilitate the well recognised phenotypic abilities of this pathogen to persist and/or grow in alkaline conditions. Alkali adaptation may be significant in the persis- tence of this pathogen in the presence of alkali detergents in food processing environments and alkali natural habitats, and has direct clinical implications in relation to virulence and response to mammalian defence mechanisms.

Scanning electron microscopy (SEM) studies revealed that exposure to 4lethal alkaline stress induced statistically significant (P

In alkaline conditions, Listeria monocytogenes cells develop higher proportions of branched-chain fatty acids (FAs), including more anteiso forms. In acid conditions, the opposite occurs. Reduced growth of pH-sensitive mutants at adverse pH (5.0/9.0) was alleviated by the addition of 2-methylbutyrate (an anteiso-FA precursor), suggesting that anteiso-FAs are important in adaptation to adverse pH. The balance between anteiso- and iso-FAs may be more important than changes in the amounts and/or degrees of saturation of FAs in pH adaptation.

Chicken Anaemia Virus (CAV) is an economically important virus that targets lymphoid and erythroblastoid progenitor cells leading to immunosuppression. This study aimed to investigate the interplay between viral infection and the host’s immune response to better understand the pathways that lead to CAV-induced immunosuppression. To mimic vertical transmission of CAV in the absence of maternally-derived antibody, day-old chicks were infected and their responses measured at various time-points post-infection by qRT-PCR and gene expression microarrays. The kinetics of mRNA expression levels of signature cytokines of innate and adaptive immune responses were determined by qRT-PCR. The global gene expression profiles of mock-infected (control) and CAV-infected chickens at 14 dpi were also compared using a chicken immune-related 5K microarray. Although in the thymus there was evidence of induction of an innate immune response following CAV infection, this was limited in magnitude. There was little evidence of a Th1 adaptive immune response in any lymphoid tissue, as would normally be expected in response to viral infection. Most cytokines associated with Th1, Th2 or Treg subsets were down-regulated, except IL-2, IL-13, IL-10 and IFNγ, which were all up-regulated in thymus and bone marrow. From the microarray studies, genes that exhibited significant (greater than 1.5-fold, false discovery rate

Background Microbiological standards within pork slaughter processing plants in the European Union are currently governed by Commission Regulation (EC) 2073/2005, which describes detailed performance criteria at specific stages of the procedure (following carcass dressing and before chilling) for total viable counts (TVC), Enterobacteriaceae (EB) and Salmonella spp. In this study, 95 carcasses from an Irish pork slaughter plant were sampled by swabbing 100 cm2 of surface at three sites (belly, ham, jowl) to examine the effects of eight processing stages (stunning, bleeding, scalding, singeing, polishing, evisceration, final inspection and chilling) on contamination levels. Results TVC ranged from approximately 1.7–6.3 log cfu cm2 during sampling. There were significant reductions in TVC for all sites after scalding and singeing (p p p Conclusions Although the bacterial numbers reported in this study may reflect specific plant practices and temporal influences, results show that contamination can be introduced at various steps in the process and highlight the importance of monitoring locations other than those required by legislation within the process. Monitoring can be used to establish baseline levels for high-risk stages specific to each plant and to assess the effectiveness of additional interventions.

Mammalian poxviruses, including vaccinia virus (VACV), have evolved multiple mechanisms to evade the host type I interferon (IFN) responses at different levels, with viral proteins targeting IFN induction, signaling, and antiviral effector functions. Avian poxviruses (avipoxviruses), which have been developed as recombinant vaccine vectors for permissive (i.e., poultry) and nonpermissive (i.e., mammals, including humans) species, encode no obvious equivalents of any of these proteins. We show that fowlpox virus (FWPV) fails to induce chicken beta IFN (ChIFN2) and is able to block its induction by transfected poly(I·C), an analog of cytoplasmic double-stranded RNA (dsRNA). A broad-scale loss-of-function genetic screen was used to find FWPV-encoded modulators of poly(I·C)-mediated ChIFN2 induction. It identified fpv012 , a member of a family of poxvirus genes highly expanded in the avipoxviruses (31 in FWPV; 51 in canarypox virus [CNPV], representing 15% of the total gene complement), encoding proteins containing N-terminal ankyrin repeats (ANKs) and C-terminal F-box-like motifs. Under ectopic expression, the first ANK of fpv012 is dispensable for inhibitory activity and the CNPV ortholog is also able to inhibit induction of ChIFN2. FWPV defective in fpv012 replicates well in culture and barely induces ChIFN2 during infection, suggesting that other factors are involved in blocking IFN induction and resisting the antiviral effectors. Nevertheless, unlike parental and revertant viruses, the mutants induce moderate levels of expression of interferon-stimulated genes (ISGs), suggesting either that there is sufficient ChIFN2 expression to partially induce the ISGs or the involvement of alternative, IFN-independent pathways that are also normally blocked by fpv012 .

Methicillin-resistant staphylococci (MRS), and methicillin-resistant Staphylococcus aureus (MRSA) in particular, have become a public and veterinary health concern. The search for MRS reservoirs outside human hospitals is needed in order to understand the reasons for their persistence and to control their spread. MRS have been isolated from rats, but little is known about their occurrence in foxes. In view of the perceived increasing proximity between people and foxes in the U.K. and the well-documented potential of foxes as hosts for zoonotic pathogens, this study examined whether foxes can be a reservoir for MRS. This study examined the carriage of staphylococci and their antimicrobial resistance patterns in 38 foxes (Vulpes vulpes) from rural and semirural areas in the U.K. Staphylococci were isolated by enrichment culture from nasal, oral, axillary, and perineal swabs and speciated by standard bacteriological tests and API ID32 STAPH (bioMerieux, Marcy l'Etoile, France). Antimicrobial resistance was investigated by disc diffusion tests and identification of mecA. Thirty-seven staphylococcal isolates were identified from 35 of the 38 foxes. All isolates were coagulase-negative and most frequently included species from the S. sciuri group (35%), S. equorum (27%), and S. capitis (22%). All were phenotypically resistant to methicillin, and mecA was detected in 33 (89%) of isolates, but only 10 (27%) showed broad β-lactam antibiotic resistance. Methicillin-resistant S. aureus was not identified. These results indicate that foxes are a potential wildlife reservoir for mecA-positive staphylococci. Selection pressure from environmental contamination with antimicrobials should be considered.

Aims Meticillin-resistant Staphylococcus aureus (MRSA) ST398 continues to spread amongst pigs and other domestic animals and man. This highlights the need for models to examine MRSA colonization and investigate control strategies. This study aimed to develop a gnotobiotic pig model and assess the potential of bacterial interference from selected coagulase-negative staphylococci (CNS) against MRSA ST398. Methods and Results Groups of 2-week-old piglets were atraumatically inoculated either with MRSA and/or CNS. Skin and mucosae were swabbed, and bacterial counts compared over a period of 21 days. Piglets developed healthily, and bacterial populations increased similarly for both MRSA and CNS until day 32. On day 37, MRSA counts in groups with CNS reduced significantly compared with MRSA alone (P = 0·03). Conclusions The results showed that inoculation of piglet skin with MRSA resulted in spontaneous colonization and that MRSA ST398 has a low pathogenic potential in gnotobiotic piglets. Quantitative bacteriology indicated that initial MRSA colonization was unaffected by concurrent CNS colonization but that interference may occur over a longer period. Significance and Impact of the Study Gnotobiotic piglets provide a reproducible model suitable for bacterial interference studies, which should be further explored as an alternative to antimicrobials in the control of MRSA.

The emergence of the livestock-associated clone of meticillin-resistant Staphylococcus aureus (MRSA) ST398 is a serious public health issue throughout Europe. In The Netherlands a stringent 'search-and-destroy' policy has been adopted, keeping low the level of MRSA prevalence. However, reports have recently emerged of transmission events between humans showing no links to livestock, contradicting belief that MRSA ST398 is poorly transmissible in humans. The question regarding the transmissibility of MRSA ST398 in humans therefore remains of great interest. Here, we investigated the capacity of MRSA ST398 to spread into an entirely susceptible human population subject to the effect of a single MRSA-positive commercial pig farm. Using a stochastic, discrete-time metapopulation model, we explored the effect of varying both the probability of persistent carriage and that of acquiring MRSA due to contact with pigs on the transmission dynamics of MRSA ST398 in humans. In particular, we assessed the value and key determinants of the basic reproduction ratio (R(0)) for MRSA ST398. Simulations showed that the presence of recurrent exposures with pigs in risky populations allows MRSA ST398 to persist in the metapopulation and transmission events to occur beyond the farming community, even when the probability of persistent carriage is low. We further showed that persistent carriage should occur in less than 10% of the time for MRSA ST398 to conserve epidemiological characteristics similar to what has been previously reported. These results indicate that implementing control policy that only targets human carriers may not be sufficient to control MRSA ST398 in the community if it remains in pigs. We argue that farm-level control measures should be implemented if an eradication programme is to be considered.

Listeria spp. can adapt to, and survive in, higher alkaline pH values than many other bacteria. This adaptation allows this important food-borne pathogen to resist subsequent (otherwise lethal) extreme alkaline stress, i.e. exposure to pH 12.0. Although such “stress hardening” may have significant public health implications by increasing the persistence of L. monocytogenes in food processing environments cleaned with alkali detergents, relatively little is known about the mechanisms and processes involved in this Alkaline–Tolerance Response (AlTR). This study examined the impact of growth phase, (alkali) pH conditions and exposure period in the extent of AlTR induction in L. monocytogenes. The extent of the AlTR (defined in terms of survival rates of adapted cells to subsequent pH 12.0 challenge) was observed to be growth-phase dependent, with significantly more cells surviving in exponential cell suspensions than in stationary cell suspensions. AlTR was induced between pH 8.0 to pH 10.0 with maximal induction (maximum cell survival) at pH 9.5. AlTR was rapidly expressed, with significantly more cells surviving in adapted suspensions (pH 9.5) for periods as short as 15 min, than in control (pH 7.4) suspensions. L. monocytogenes displayed different alkali responses on exposure to different types of alkali. These results confirm the hypothesis that L. monocytogenes responds to alkali pH through a complex network of responses that result to a phenotypic gain of resistance depending on many biological variables. In view of the fact that alkaline conditions are commonly encountered in food processing/preservation and clinical environments, these responses have profound implications to public health.

Staphylococcus aureus is a major community and nosocomial pathogen. Its ability to withstand multiple stress conditions and quickly develop resistance to antibiotics complicates the control of staphylococcal infections. Adaptation to lower temperatures is a key for the survival of bacterial species outside the host. Branched-chain α-keto acid dehydrogenase (BKD) is an enzyme complex that catalyzes the early stages of branched-chain fatty acid (BCFA) production. In this study, BKD was inactivated, resulting in reduced levels of BCFAs in the membrane of S. aureus . Growth of the BKD-inactivated mutant was progressively more impaired than that of wild-type S. aureus with decreasing temperature, to the point that the mutant could not grow at 12°C. The growth of the mutant was markedly stimulated by the inclusion of 2-methylbutyrate in the growth medium at all temperatures tested. 2-Methylbutyrate is a precursor of odd-numbered anteiso fatty acids and bypasses BKD. Interestingly, growth of wild-type S. aureus was also stimulated by including 2-methylbutyrate in the medium, especially at lower temperatures. The anteiso fatty acid content of the BKD-inactivated mutant was restored by the inclusion of 2-methylbutyrate in the medium. Fluorescence polarization measurements indicated that the membrane of the BKD-inactivated mutant was significantly less fluid than that of wild-type S. aureus . Consistent with this result, the mutant showed decreased toluene tolerance that could be increased by the inclusion of 2-methylbutyrate in the medium. The BKD-inactivated mutant was more susceptible to alkaline pH and oxidative stress conditions. Inactivation of the BKD enzyme complex in S. aureus also led to a reduction in adherence of the mutant to eukaryotic cells and its survival in a mouse host. In addition, the mutant offers a tool to study the role of membrane fluidity in the interaction of S. aureus with antimicrobial substances.

Many of the considerable abilities of Listeria monocytogenes to persist and grow in a wide range of adverse environmental conditions are thought to be at least partly under the control of the alternative sigma factor (sigma(B)), encoded by the sigB gene. However, little is known about the role of this master regulon in the impressive ability of Listeria to persist and grow under conditions of alkaline pH. In this study, Northern blot analysis of parent Listeria mRNA revealed that alkali adaptation (pH 9.5 for 1 h) significantly increased the expression of sigB-derived mRNA. The study included a comparison of the relative survival of mid-exponential populations of adapted and nonadapted parent type (sigma(B) expressing) and mutant (not (TB expressing, Delta sigB) Listeria strains during subsequent alkaline (pH 12.0), osmotic (25% NaCl, wt/vol), or ethanol (16.5%) stress. Alkali-adapted parent strains were more resistant to pH 12.0 than were adapted Delta sigB type strains, but both alkali-adapted parent and Delta sigB strains were more resistant to pH 12.0 than were nonadapted strains. Alkali-adapted parent strains were more resistant to osmotic stress than were adapted Delta sigB type strains. No significant differences in viability were observed between alkali-adapted parent and Delta sigB strains after ethanol stress, suggesting that cross-protection against osmotic stress is mediated by sigma(B) whereas cross-protection against ethanol is sigma(B) independent. Overall, alkali-induced cross-protection against osmotic and ethanol challenges may have serious implications for food safety and human health because such stress conditions are routinely used as part of food preservation and surface cleaning processes.

Chicken, which is almost universally-acceptable, will shortly become the most consumed meat in the developed and developing world. Viruses of chickens, whether enzootic (e.g. Newcastle, Mareks or infectious bursal disease viruses or infectious bronchitis virus) or zoonotic (especially avian influenza viruses such as H5N1 and H7N9), pose major threats to the supply of this globally important source of protein. Understanding the nature of the chicken innate responses will help us better understand these viruses, their evolution and their pathogenesis, and may help us develop more effective methods of controlling them. Although first discovered in chicken cells, details of the chicken interferon response are only just being elucidated, because chicken interferon was only cloned in 1994 and the chicken genome sequence was only reported in 2004. It is noteworthy that the identity of interferon-stimulated genes is far better described in mammals. Although there are increasing numbers of reports of chicken responses to virus infection, most of the regulated genes described are involved in the cell cycle, metabolic pathways and cellular structures required for virus replication. We have conducted a study aimed at describing the complement of potential anti-viral genes, identifying those induced by recombinant type I interferon in a common research and vaccine production substrate, namely primary chick embryo fibroblasts. The results are robust because 155 of the 193 genes induced were detected by at least two of the three transcriptomic technologies employed: Illumina RNA-seq, the Affymetrix 32K GeneChip Chicken Genome Array and the Chicken Gene 1.0 ST Array.

We conducted microarray analysis of the increasingly popular chicken DF-1 cell line and its primary progenitor, chicken embryo fibroblast cells (CEF), in both normal and stimulated conditions using recombinant chicken IFN- α and the CEF-adapted infectious bursal disease virus vaccine strain PBG98. We found that DF-1 cells have an attenuated innate immune response compared to CEF, and also that chicken SOCS1 (ChSOCS1), a negative regulator of cytokine signaling, is expressed at levels 13-fold higher in DF-1 cells than in CEF. We found that overexpressing ChSOCS1 in CEF reduced levels of phosphorylated STAT1 protein in response to viral infection but that its “SOCS box protein” domain (which, in mammalian SOCS1, interacts with an E3 ubiquitin ligase complex) is not essential for the inhibition of cytokine-induced JAK/STAT signaling activation in DF-1 cells. Overexpression of SOCS1 in DF-1 cells led to a significant relative decrease in expression of IFN- β , MX1, IFIT5 and MDA5 in response to PBG98 infection, and increased viral yield. Conversely, knockdown of SOCS1 increased ISG induction and reduced viral yield in IFN-stimulated DF-1 cells. Our results show that, like its mammalian counterpart, ChSOCS1 reduces induction of the IFN signaling pathway.