Profiling Host MicroRNA Responses to Henipavirus Infection.

Diseases caused by henipaviruses feature incubation periods of up to 16 days, during which infected animals may show no apparent signs of disease yet be capable of transmitting the virus to humans. This risk has prompted research into host-derived biomarkers for early disease detection. Here, we describe a methodology for the assaying of host microRNAs (miRs), small non-coding RNAs that show promise as biomarkers for several human diseases and are responsive during early-stage henipavirus infection. In addition to their potential as disease biomarkers, miRNA profiling of henipavirus infections provides insight into cellular and immune pathways associated with disease pathogenesis.

Identification of a novel role for the immunomodulator ILRUN in the development of several T cell subsets in mice.

Inflammation and lipid regulator with UBA-like and NBR1-like domains (ILRUN) is a protein-encoding gene associated with innate immune signaling, lipid metabolism and cancer. In the context of innate immunity, ILRUN inhibits IRF3-mediated transcription of antimicrobial and proinflammatory cytokines by inducing degradation of the transcriptional coactivators CBP and p300. There remains a paucity of information, however, regarding the innate immune roles of ILRUN beyond in vitro analyses. To address this, we utilize a knockout mouse model to investigate the effect of ILRUN on cytokine expression in splenocytes and on the development of immune cell populations in the spleen and thymus. We show elevated production of tumor necrosis factor and interleukin-6 cytokines in ILRUN-deficient splenocytes following stimulation with the innate immune ligands polyinosinic:polycytidylic acid or lipopolysaccharide. Differences were also observed in the populations of several T cell subsets, including regulatory, mucosal-associated invariant and natural killer. These data identify novel functions for ILRUN in the development of certain immune cell populations and support previous in vitro findings that ILRUN negatively regulates the synthesis of pathogen-stimulated cytokines. This establishes the ILRUN knockout mouse model as a valuable resource for further study of the functions of ILRUN in health and disease.

Germline engineering of the chicken genome using CRISPR/Cas9 by in vivo transfection of PGCs.

Development of simple and readily adoptable methods to mediate germline engineering of the chicken genome will have many applications in research, agriculture and industrial biotechnology. We report germline targeting of the endogenous chicken Interferon Alpha and Beta Receptor Subunit 1 (IFNAR1) gene by in vivo transgenic expression of the high-fidelity Cas9 (Cas9-HF1) and guide RNAs (gRNAs) in chickens. First, we developed a Tol2 transposon vector carrying Cas9-HF1, IFNAR1-gRNAs (IF-gRNAs) and green fluorescent protein (GFP) transgenes (pTgRCG) and validated in chicken fibroblast DF1 cells. Next, the pTgRCG plasmid was directly injected into the dorsal aorta of embryonic day (ED) 2.5 chicken embryos targeting the circulating primordial germ cells (PGCs). The resulting chimera roosters generated a fully transgenic generation 1 (G1) hen with constitutive expression of Cas9-HF1 and IF-gRNAs (G1_Tol2-Cas9/IF-gRNA). We detected a spectrum of indels at gRNA-targeted loci in the G1_Tol2-Cas9/IF-gRNA hen and the indels were stably inherited by the G2 progeny. Breeding of the G1_Tol2-Cas9/IF-gRNA hen resulted in up to 10% transgene-free heterozygote IFNAR1 mutants, following null-segregation of the Tol2 insert. The method described here will provide new opportunities for genome editing in chicken and other avian species that lack PGC culture.

H7N9 bearing a mutation in the nucleoprotein leads to increased pathology in chickens.

The zoonotic H7N9 avian influenza (AI) virus first emerged in 2013 as a low pathogenic (LPAI) strain, and has repeatedly caused human infection resulting in severe respiratory illness and a mortality of ~39% (>600 deaths) across five epidemic waves. This virus has circulated in poultry with little to no discernible clinical signs, making detection and control difficult. Contrary to published data, our group has observed a subset of specific pathogen free chickens infected with the H7N9 virus succumb to disease, showing clinical signs consistent with highly pathogenic AI (HPAI). Viral genome sequencing revealed two key mutations had occurred following infection in the haemagglutinin (HA 226 L>Q) and nucleoprotein (NP 373 A>T) proteins. We further investigated the impact of the NP mutation and demonstrated that only chickens bearing a single nucleotide polymorphism (SNP) in their IFITM1 gene were susceptible to the H7N9 virus. Susceptible chickens demonstrated a distinct loss of CD8+ T cells from the periphery as well as a dysregulation of IFNγ that was not observed for resistant chickens, suggesting a role for the NP mutation in altered T cell activation. Alternatively, it is possible that this mutation led to altered polymerase activity, as the mutation occurs in the NP 360-373 loop which has been previously show to be important in RNA binding. These data have broad ramifications for our understanding of the pathobiology of AI in chickens and humans and provide an excellent model for investigating the role of antiviral genes in a natural host species.

Interferon Signaling in Chickens Plays a Crucial Role in Inhibiting Influenza Replication in DF1 Cells.

Influenza A viruses (IAV) pose a constant threat to human and poultry health. Of particular interest are the infections caused by highly pathogenic avian influenza (HPAI) viruses, such as H5N1, which cause significant production issues. In response to influenza infection, cells activate immune mechanisms that lead to increased interferon (IFN) production. To investigate how alterations in the interferon signaling pathway affect the cellular response to infection in the chicken, we used CRISPR/Cas9 to generate a chicken cell line that lacks a functional the type I interferon receptor (IFNAR1). We then assessed viral infections with the WSN strain of influenza. Cells lacking a functional IFNAR1 receptor showed reduced expression of the interferon stimulated genes (ISG) such as Protein Kinase R (PKR) and Myxovirus resistance (Mx) and were more susceptible to viral infection with WSN. We further investigated the role or IFNAR1 on low pathogenicity avian influenza (LPAI) strains (H7N9) and a HPAI strain (H5N1). Intriguingly, Ifnar-/- cells appeared more resistant than WT cells when infected with HPAI virus, potentially indicating a different interaction between H5N1 and the IFN signaling pathway. Our findings support that ChIFNAR1 is a key component of the chicken IFN signaling pathway and these data add contributions to the field of host-avian pathogen interaction and innate immunity in chickens.

Ferret Interferon (IFN)-Inducible Transmembrane Proteins Are Upregulated by both IFN-α and Influenza Virus Infection.

The current fears of a future influenza pandemic have resulted in an increased emphasis on the development and testing of novel therapeutic strategies against the virus. Fundamental to this is the ferret model of influenza infection, which is critical in examining pathogenesis and treatment. Nevertheless, a precise evaluation of the efficacy of any treatment strategy in ferrets is reliant on understanding the immune response in this model. Interferon-inducible transmembrane proteins (IFITMs) are interferon-stimulated proteins shown to be critically important in the host immune response against viral infections. These proteins confer intrinsic innate immunity to pH-dependent viruses such as influenza viruses and can inhibit cytosolic entry of such viruses to limit the severity of infection following interferon upregulation. Mutations in IFITM genes in humans have been identified as key risk factors for worsened disease progression, particularly in the case of avian influenza viruses such as H7N9. While the IFITM genes of humans and mice have been well characterized, no studies have been conducted to classify the IFITM locus and interferon-driven upregulation of IFITMs in ferrets. Here, we show the architecture of the ferret IFITM locus and its synteny to the IFITM locus of other mammalian and avian species. Furthermore, we show that ferret IFITM1, -2, and -3 are functionally responsive to both interferon-α (IFN-α) and influenza virus stimulation. Thus, we show that ferret IFITMs exhibit interferon-stimulated properties similar to those shown in other species, furthering our knowledge of the innate immune response in the ferret model of human influenza virus infections. IMPORTANCE IFITM proteins can prevent the entry of several pH-dependent viruses, including high-consequence viruses such as HIV, influenza viruses, and SARS-coronaviruses. Mutations in these genes have been associated with worsened disease outcomes with mutations in their IFITM genes, highlighting these genes as potential disease risk factors. Ferrets provide a valuable tool to model infectious diseases; however, there is a critical shortage of information regarding their interferon-stimulated genes. We identified the putative ferret IFITM genes and mapped their complete gene locus. Thus, our study fills a critical gap in knowledge and supports the further use of the ferret model to explore the importance of IFITMs in these important diseases.

ILRUN Downregulates ACE2 Expression and Blocks Infection of Human Cells by SARS-CoV-2.

The human protein-coding gene ILRUN (inflammation and lipid regulator with UBA-like and NBR1-like domains; previously C6orf106) was identified as a proviral factor for Hendra virus infection and was recently characterized to function as an inhibitor of type I interferon expression. Here, we have utilized transcriptome sequencing (RNA-seq) to define cellular pathways regulated by ILRUN in the context of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of Caco-2 cells. We find that inhibition of ILRUN expression by RNA interference alters transcription profiles of numerous cellular pathways, including upregulation of the SARS-CoV-2 entry receptor ACE2 and several other members of the renin-angiotensin aldosterone system. In addition, transcripts of the SARS-CoV-2 coreceptors TMPRSS2 and CTSL were also upregulated. Inhibition of ILRUN also resulted in increased SARS-CoV-2 replication, while overexpression of ILRUN had the opposite effect, identifying ILRUN as a novel antiviral factor for SARS-CoV-2 replication. This represents, to our knowledge, the first report of ILRUN as a regulator of the renin-angiotensin-aldosterone system (RAAS). IMPORTANCE There is no doubt that the current rapid global spread of COVID-19 has had significant and far-reaching impacts on our health and economy and will continue to do so. Research in emerging infectious diseases, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is growing rapidly, with new breakthroughs in the understanding of host-virus interactions to assist with the development of innovative and exciting therapeutic strategies. Here, we present the first evidence that modulation of the human protein-coding gene ILRUN functions as an antiviral factor for SARS-CoV-2 infection, likely through its newly identified role in regulating the expression of SARS-CoV-2 entry receptors ACE2, TMPRSS2, and CTSL. These data improve our understanding of biological pathways that regulate host factors critical to SARS-CoV-2 infection, contributing to the development of antiviral strategies to deal with the current SARS-CoV-2 pandemic.

Ribosome-Profiling Reveals Restricted Post Transcriptional Expression of Antiviral Cytokines and Transcription Factors during SARS-CoV-2 Infection.

The global COVID-19 pandemic caused by SARS-CoV-2 has resulted in over 2.2 million deaths. Disease outcomes range from asymptomatic to severe with, so far, minimal genotypic change to the virus so understanding the host response is paramount. Transcriptomics has become incredibly important in understanding host-pathogen interactions; however, post-transcriptional regulation plays an important role in infection and immunity through translation and mRNA stability, allowing tight control over potent host responses by both the host and the invading virus. Here, we apply ribosome profiling to assess post-transcriptional regulation of host genes during SARS-CoV-2 infection of a human lung epithelial cell line (Calu-3). We have identified numerous transcription factors (JUN, ZBTB20, ATF3, HIVEP2 and EGR1) as well as select antiviral cytokine genes, namely IFNB1, IFNL1,2 and 3, IL-6 and CCL5, that are restricted at the post-transcriptional level by SARS-CoV-2 infection and discuss the impact this would have on the host response to infection. This early phase restriction of antiviral transcripts in the lungs may allow high viral load and consequent immune dysregulation typically seen in SARS-CoV-2 infection.

Investigating the Interaction between Negative Strand RNA Viruses and Their Hosts for Enhanced Vaccine Development and Production.

The current pandemic has highlighted the ever-increasing risk of human to human spread of zoonotic pathogens. A number of medically-relevant zoonotic pathogens are negative-strand RNA viruses (NSVs). NSVs are derived from different virus families. Examples like Ebola are known for causing severe symptoms and high mortality rates. Some, like influenza, are known for their ease of person-to-person transmission and lack of pre-existing immunity, enabling rapid spread across many countries around the globe. Containment of outbreaks of NSVs can be difficult owing to their unpredictability and the absence of effective control measures, such as vaccines and antiviral therapeutics. In addition, there remains a lack of essential knowledge of the host-pathogen response that are induced by NSVs, particularly of the immune responses that provide protection. Vaccines are the most effective method for preventing infectious diseases. In fact, in the event of a pandemic, appropriate vaccine design and speed of vaccine supply is the most critical factor in protecting the population, as vaccination is the only sustainable defense. Vaccines need to be safe, efficient, and cost-effective, which is influenced by our understanding of the host-pathogen interface. Additionally, some of the major challenges of vaccines are the establishment of a long-lasting immunity offering cross protection to emerging strains. Although many NSVs are controlled through immunisations, for some, vaccine design has failed or efficacy has proven unreliable. The key behind designing a successful vaccine is understanding the host-pathogen interaction and the host immune response towards NSVs. In this paper, we review the recent research in vaccine design against NSVs and explore the immune responses induced by these viruses. The generation of a robust and integrated approach to development capability and vaccine manufacture can collaboratively support the management of outbreaking NSV disease health risks.

The Dynamics of the Ferret Immune Response During H7N9 Influenza Virus Infection.

As the recent outbreak of SARS-CoV-2 has highlighted, the threat of a pandemic event from zoonotic viruses, such as the deadly influenza A/H7N9 virus subtype, continues to be a major global health concern. H7N9 virus strains appear to exhibit greater disease severity in mammalian hosts compared to natural avian hosts, though the exact mechanisms underlying this are somewhat unclear. Knowledge of the H7N9 host-pathogen interactions have mainly been constrained to natural sporadic human infections. To elucidate the cellular immune mechanisms associated with disease severity and progression, we used a ferret model to closely resemble disease outcomes in humans following influenza virus infection. Intriguingly, we observed variable disease outcomes when ferrets were inoculated with the A/Anhui/1/2013 (H7N9) strain. We observed relatively reduced antigen-presenting cell activation in lymphoid tissues which may be correlative with increased disease severity. Additionally, depletions in CD8+ T cells were not apparent in sick animals. This study provides further insight into the ways that lymphocytes maturate and traffic in response to H7N9 infection in the ferret model.

Molecular characterisation of ILRUN, a novel inhibitor of proinflammatory and antimicrobial cytokines.

Regulation of type-I interferon (IFN) production is essential to the balance between antimicrobial defence and autoimmune disorders. The human protein-coding gene ILRUN (inflammation and lipid regulator with UBA-like and NBR1-like domains, previously C6orf106) was recently characterised as an inhibitor of antiviral and proinflammatory cytokine (interferon-alpha/beta and tumor necrosis factor alpha) transcription. Currently there is a paucity of information about the molecular characteristics of ILRUN, despite it being associated with several diseases including virus infection, coronary artery disease, obesity and cancer. Here, we characterise ILRUN as a highly phylogenetically conserved protein containing UBA-like and a NBR1-like domains that are both essential for inhibition of type-I interferon and tumor necrosis factor alpha) transcription in human cells. We also solved the crystal structure of the NBR1-like domain, providing insights into its potential role in ILRUN function. This study provides critical information for future investigations into the role of ILRUN in health and disease.

MicroRNA Biomarkers for Infectious Diseases: From Basic Research to Biosensing.

In the pursuit of improved diagnostic tests for infectious diseases, several classes of molecules have been scrutinized as prospective biomarkers. Small (18-22 nucleotide), non-coding RNA transcripts called microRNAs (miRNAs) have emerged as promising candidates with extensive diagnostic potential, due to their role in numerous diseases, previously established methods for quantitation and their stability within biofluids. Despite efforts to identify, characterize and apply miRNA signatures as diagnostic markers in a range of non-infectious diseases, their application in infectious disease has advanced relatively slowly. Here, we outline the benefits that miRNA biomarkers offer to the diagnosis, management, and treatment of infectious diseases. Investigation of these novel biomarkers could advance the use of personalized medicine in infectious disease treatment, which raises important considerations for validating their use as diagnostic or prognostic markers. Finally, we discuss new and emerging miRNA detection platforms, with a focus on rapid, point-of-care testing, to evaluate the benefits and obstacles of miRNA biomarkers for infectious disease.

Correction: ADAMTS5 Is a Critical Regulator of Virus-Specific T Cell Immunity.

[This corrects the article DOI: 10.1371/journal.pbio.1002580.].

Extreme Competence: Keystone Hosts of Infections.

Individual hosts differ extensively in their competence for parasites, but traditional research has discounted this variation, partly because modeling such heterogeneity is difficult. This discounting has diminished as tools have improved and recognition has grown that some hosts, the extremely competent, can have exceptional impacts on disease dynamics. Most prominent among these hosts are the superspreaders, but other forms of extreme competence (EC) exist and others await discovery; each with potentially strong but distinct implications for disease emergence and spread. Here, we propose a framework for the study and discovery of EC, suitable for different host-parasite systems, which we hope enhances our understanding of how parasites circulate and evolve in host communities.

The Drivers of Pathology in Zoonotic Avian Influenza: The Interplay Between Host and Pathogen.

The emergence of zoonotic strains of avian influenza (AI) that cause high rates of mortality in people has caused significant global concern, with a looming threat that one of these strains may develop sustained human-to-human transmission and cause a pandemic outbreak. Most notable of these viral strains are the H5N1 highly pathogenic AI and the H7N9 low pathogenicity AI viruses, both of which have mortality rates above 30%. Understanding of their mechanisms of infection and pathobiology is key to our preparation for these and future viral strains of high consequence. AI viruses typically circulate in wild bird populations, commonly infecting waterfowl and also regularly entering commercial poultry flocks. Live poultry markets provide an ideal environment for the spread AI and potentially the selection of mutants with a greater propensity for infecting humans because of the potential for spill over from birds to humans. Pathology from these AI virus infections is associated with a dysregulated immune response, which is characterized by systemic spread of the virus, lymphopenia, and hypercytokinemia. It has been well documented that host/pathogen interactions, particularly molecules of the immune system, play a significant role in both disease susceptibility as well as disease outcome. Here, we review the immune/virus interactions in both avian and mammalian species, and provide an overview or our understanding of how immune dysregulation is driven. Understanding these susceptibility factors is critical for the development of new vaccines and therapeutics to combat the next pandemic influenza.

C6orf106 is a novel inhibitor of the interferon-regulatory factor 3-dependent innate antiviral response.

Host recognition of intracellular viral RNA and subsequent induction of cytokine signaling are tightly regulated at the cellular level and are a target for manipulation by viruses and therapeutics alike. Here, we characterize chromosome 6 ORF 106 (C6orf106) as an evolutionarily conserved inhibitor of the innate antiviral response. C6orf106 suppresses the synthesis of interferon (IFN)-α/ß and proinflammatory tumor necrosis factor (TNF) α in response to the dsRNA mimic poly(I:C) and to Sendai virus infection. Unlike canonical inhibitors of antiviral signaling, C6orf106 blocks interferon-regulatory factor 3 (IRF3) and, to a lesser extent, NF-κB activity without modulating their activation, nuclear translocation, cellular expression, or degradation. Instead, C6orf106 interacts with IRF3 and inhibits IRF3 recruitment to type I IFN promoter sequences while also reducing the nuclear levels of the coactivator proteins p300 and CREB-binding protein (CBP). In summary, we have defined C6orf106 as a negative regulator of antiviral immunity that blocks IRF3-dependent cytokine production via a noncanonical and poorly defined mechanism. This work presents intriguing implications for antiviral immunity, autoimmune disorders, and cancer.

A Functional Genomics Approach to Henipavirus Research: The Role of Nuclear Proteins, MicroRNAs and Immune Regulators in Infection and Disease.

Hendra and Nipah viruses (family Paramyxoviridae, genus Henipavirus) are zoonotic RNA viruses that cause lethal disease in humans and are designated as Biosafety Level 4 (BSL4) agents. Moreover, henipaviruses belong to the same group of viruses that cause disease more commonly in humans such as measles, mumps and respiratory syncytial virus. Due to the relatively recent emergence of the henipaviruses and the practical constraints of performing functional genomics studies at high levels of containment, our understanding of the henipavirus infection cycle is incomplete. In this chapter we describe recent loss-of-function (i.e. RNAi) functional genomics screens that shed light on the henipavirus-host interface at a genome-wide level. Further to this, we cross-reference RNAi results with studies probing host proteins targeted by henipavirus proteins, such as nuclear proteins and immune modulators. These functional genomics studies join a growing body of evidence demonstrating that nuclear and nucleolar host proteins play a crucial role in henipavirus infection. Furthermore these studies will underpin future efforts to define the role of nucleolar host-virus interactions in infection and disease.

Circulating microRNA profiles of Hendra virus infection in horses.

Hendra virus (HeV) is an emerging zoonotic pathogen harbored by Australian mainland flying foxes. HeV infection can cause lethal disease in humans and horses, and to date all cases of human HeV disease have resulted from contact with infected horses. Currently, diagnosis of acute HeV infections in horses relies on the productive phase of infection when virus shedding may occur. An assay that identifies infected horses during the preclinical phase of infection would reduce the risk of zoonotic viral transmission during management of HeV outbreaks. Having previously shown that the host microRNA (miR)-146a is upregulated in the blood of HeV-infected horses days prior to the detection of viremia, we have profiled miRNAs at the transcriptome-wide level to comprehensively assess differences between infected and uninfected horses. Next-generation sequencing and the miRDeep2 algorithm identified 742 mature miRNA transcripts corresponding to 593 miRNAs in whole blood of six horses (three HeV-infected, three uninfected). Thirty seven miRNAs were differentially expressed in infected horses, two of which were validated by qRT-PCR. This study describes a methodology for the transcriptome-wide profiling of miRNAs in whole blood and supports the notion that measuring host miRNA expression levels may aid infectious disease diagnosis in the future.

Breaking the chain of zoonoses through biosecurity in livestock.

Increases in global travel, trade and urbanisation are leading to greater incidence of zoonotic disease, and livestock are often a key link in the spread of disease to humans. As such, livestock vaccination strategies, as a part of broader biosecurity solutions, are critical to both animal and human health. Importantly, approaches that restrict infectious agents in livestock, not only protects their economic value but should reduce the potential for spill over infections in humans. Biosecurity solutions to livestock health can take a number of different forms and are generally heavily weighted towards prevention of infection rather than treatment. Therefore, vaccination can provide an effective component of a strategic approach, particularly as production economics dictate the use of cost effective solutions. Furthermore, in an evolving global environment there is a need for vaccines that accommodate for lower socioeconomic and rapidly emerging zoonotics.

Development of an anti-ferret CD4 monoclonal antibody for the characterisation of ferret T lymphocytes.

The ferret is an established animal model for a number of human respiratory viral infections, such as influenza virus and more recently, Ebola virus. However, a paucity of immunological reagents has hampered the study of cellular immune responses. Here we describe the development and characterisation of a novel monoclonal antibody (mAb) against the ferret CD4 antigen and the characterisation of ferret CD4 T lymphocytes. Recombinant production and purification of the ferret CD4 ectodomain soluble protein allowed hybridoma generation and the generation of a mAb (FeCD4) showing strong binding to ferret CD4 protein and lymphoid cells by flow cytometry. FeCD4 bound to its cognate antigen post-fixation with paraformaldehyde (PFA) which is routinely used to inactivate highly pathogenic viruses. We have also used FeCD4 in conjunction with other immune cell markers to characterise ferret T cells in both primary and secondary lymphoid organs. In summary, we have developed an important reagent for the study of cellular immunological responses in the ferret model of infectious disease.