The antiviral potential of the antiandrogen enzalutamide and the viral-androgen signaling interplay in seasonal coronaviruses.

The sex disparity in COVID-19 outcomes with males generally faring worse than females has been associated with the androgen-regulated expression of the protease TMPRSS2 and the cell receptor ACE2 in the lung and fueled interest in antiandrogens as potential antivirals. In this study, we explored enzalutamide, an antiandrogen used commonly to treat prostate cancer, as a potential antiviral against the human coronaviruses which cause seasonal respiratory infections (HCoV-NL63, -229E, and -OC43). Using lentivirus-pseudotyped and authentic HCoV, we report that enzalutamide reduced 229E and NL63 entry and infection in both TMPRSS2- and nonexpressing immortalized cells, suggesting a TMPRSS2-independent mechanism. However, no effect was observed against OC43. To decipher this distinction, we performed RNA-sequencing analysis on 229E- and OC43-infected primary human airway cells. Our results show a significant induction of androgen-responsive genes by 229E compared to OC43 at 24 and 72 h postinfection. The virus-mediated effect on AR-signaling was further confirmed with a consensus androgen response element-driven luciferase assay in androgen-depleted MRC-5 cells. Specifically, 229E induced luciferase-reporter activity in the presence and absence of the synthetic androgen mibolerone, while OC43 inhibited induction. These findings highlight a complex interplay between viral infections and androgen-signaling, offering insights for disparities in viral outcomes and antiviral interventions.

Serological evidence of virus infection in Eidolon helvum fruit bats: implications for bushmeat consumption in Nigeria.

Introduction: The Eidolon helvum fruit bat is one of the most widely distributed fruit bats in Africa and known to be a reservoir for several pathogenic viruses that can cause disease in animals and humans. To assess the risk of zoonotic spillover, we conducted a serological survey of 304 serum samples from E. helvum bats that were captured for human consumption in Makurdi, Nigeria. Methods: Using pseudotyped viruses, we screened 304 serum samples for neutralizing antibodies against viruses from the Coronaviridae, Filoviridae, Orthomyxoviridae and Paramyxoviridae families. Results: We report the presence of neutralizing antibodies against henipavirus lineage GH-M74a virus (odds ratio 6.23; p < 0.001), Nipah virus (odds ratio 4.04; p = 0.00031), bat influenza H17N10 virus (odds ratio 7.25; p < 0.001) and no significant association with Ebola virus (odds ratio 0.56; p = 0.375) in this bat cohort. Conclusion: The data suggest a potential risk of zoonotic spillover including the possible circulation of highly pathogenic viruses in E. helvum populations. These findings highlight the importance of maintaining sero-surveillance of E. helvum, and the necessity for further, more comprehensive investigations to monitor changes in virus prevalence, distribution over time, and across different geographic locations.

Use of Antiandrogens as Therapeutic Agents in COVID-19 Patients.

COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), is estimated to have caused over 6.5 million deaths worldwide. The emergence of fast-evolving SARS-CoV-2 variants of concern alongside increased transmissibility and/or virulence, as well as immune and vaccine escape capabilities, highlight the urgent need for more effective antivirals to combat the disease in the long run along with regularly updated vaccine boosters. One of the early risk factors identified during the COVID-19 pandemic was that men are more likely to become infected by the virus, more likely to develop severe disease and exhibit a higher likelihood of hospitalisation and mortality rates compared to women. An association exists between SARS-CoV-2 infectiveness and disease severity with sex steroid hormones and, in particular, androgens. Several studies underlined the importance of the androgen-mediated regulation of the host protease TMPRSS2 and the cell entry protein ACE2, as well as the key role of these factors in the entry of the virus into target cells. In this context, modulating androgen signalling is a promising strategy to block viral infection, and antiandrogens could be used as a preventative measure at the pre- or early hospitalisation stage of COVID-19 disease. Different antiandrogens, including commercial drugs used to treat metastatic castration-sensitive prostate cancer and other conditions, have been tested as antivirals with varying success. In this review, we summarise the most recent updates concerning the use of antiandrogens as prophylactic and therapeutic options for COVID-19.

Naïve Human Macrophages Are Refractory to SARS-CoV-2 Infection and Exhibit a Modest Inflammatory Response Early in 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 exposed MDM with notable absence of IFN-ß induction, which was instead strongly induced by the influenza viruses. Pre-treatment of MDM with IFN-α enhanced proinflammatory cytokine expression upon exposure to virus. Together, the findings concur that the hyperinflammation observed in SARS-CoV-2 infection is not driven by macrophages.

Transcriptomic Analysis of Inbred Chicken Lines Reveals Infectious Bursal Disease Severity 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.

Modulation of Early Host Innate Immune Response by an Avipox Vaccine Virus' Lateral Body Protein.

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

Author Correction: Hypoxic gene expression in chronic hepatitis B virus infected patients is not observed in state-of-the-art in vitro and mouse infection models.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Hypoxic gene expression in chronic hepatitis B virus infected patients is not observed in state-of-the-art in vitro and mouse infection models.

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.

Inferring the Urban Transmission Potential of Bat Influenza Viruses.

Bats are considered natural reservoirs of various, potentially zoonotic viruses, exemplified by the influenza A-like viruses H17N10 and H18N11 in asymptomatic Neotropical bats. These influenza viruses are evolutionarily distinct, are poorly adapted to laboratory mice and ferrets and cannot reassort in vitro with conventional strains to form new influenza subtypes. However, they have attracted renewed attention following reports that their entry in host cells is mediated by the trans-species conserved MHC-II proteins, suggesting that they hold zoonotic potential. Despite the recent studies, the viruses' epidemiology and public health significance remain incompletely understood. Delineating the mechanistic basis of the interactions with their hosts and assessing their global distribution are essential in order to fully assess the zoonotic threat that these strains pose.

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 IFNα, IFNß, 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-ß 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 cGAS Senses Fowlpox Virus Infection and Regulates Macrophage Effector Functions.

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 signaling 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.

Entry of the bat influenza H17N10 virus into mammalian cells is enabled by the MHC class II HLA-DR receptor.

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.

Chicken Embryonic-Stem Cells Are Permissive to Poxvirus Recombinant Vaccine Vectors.

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., NFκB, 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.

Spotlight on avian pathology: fowlpox virus.

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.

Chicken anaemia virus evades host immune responses in transformed lymphocytes.

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.

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 signalling (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.

An Online Survey on Consumer Knowledge and Understanding of Added Sugars.

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.

Chicken interferome: avian interferon-stimulated genes identified by microarray and RNA-seq of primary chick embryo fibroblasts treated with a chicken type I interferon (IFN-α).

Viruses that infect birds pose major threats-to the global supply of chicken, the major, universally-acceptable meat, and as zoonotic agents (e.g. avian influenza viruses H5N1 and H7N9). Controlling these viruses in birds as well as understanding their emergence into, and transmission amongst, humans will require considerable ingenuity and understanding of how different species defend themselves. The type I interferon-coordinated response constitutes the major antiviral innate defence. Although interferon was discovered in chicken cells, details of the response, particularly the identity of hundreds of stimulated genes, are far better described in mammals. Viruses induce interferon-stimulated genes but they also regulate the expression of many hundreds of cellular metabolic and structural genes to facilitate their replication. This study focusses on the potentially anti-viral genes by identifying those induced just by interferon in primary chick embryo fibroblasts. Three transcriptomic technologies were exploited: RNA-seq, a classical 3'-biased chicken microarray and a high density, "sense target", whole transcriptome chicken microarray, with each recognising 120-150 regulated genes (curated for duplication and incorrect assignment of some microarray probesets). Overall, the results are considered robust because 128 of the compiled, curated list of 193 regulated genes were detected by two, or more, of the technologies.

Species difference in ANP32A underlies influenza A virus polymerase host restriction.

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.