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.

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.

Detection of SARS-CoV-2 infection by microRNA profiling of the upper respiratory tract.

Host biomarkers are increasingly being considered as tools for improved COVID-19 detection and prognosis. We recently profiled circulating host-encoded microRNA (miRNAs) during SARS-CoV-2 infection, revealing a signature that classified COVID-19 cases with 99.9% accuracy. Here we sought to develop a signature suited for clinical application by analyzing specimens collected using minimally invasive procedures. Eight miRNAs displayed altered expression in anterior nasal tissues from COVID-19 patients, with miR-142-3p, a negative regulator of interleukin-6 (IL-6) production, the most strongly upregulated. Supervised machine learning analysis revealed that a three-miRNA signature (miR-30c-2-3p, miR-628-3p and miR-93-5p) independently classifies COVID-19 cases with 100% accuracy. This study further defines the host miRNA response to SARS-CoV-2 infection and identifies candidate biomarkers for improved COVID-19 detection.

Altered microRNA expression in COVID-19 patients enables identification of SARS-CoV-2 infection.

The host response to SARS-CoV-2 infection provide insights into both viral pathogenesis and patient management. The host-encoded microRNA (miRNA) response to SARS-CoV-2 infection, however, remains poorly defined. Here we profiled circulating miRNAs from ten COVID-19 patients sampled longitudinally and ten age and gender matched healthy donors. We observed 55 miRNAs that were altered in COVID-19 patients during early-stage disease, with the inflammatory miR-31-5p the most strongly upregulated. Supervised machine learning analysis revealed that a three-miRNA signature (miR-423-5p, miR-23a-3p and miR-195-5p) independently classified COVID-19 cases with an accuracy of 99.9%. In a ferret COVID-19 model, the three-miRNA signature again detected SARS-CoV-2 infection with 99.7% accuracy, and distinguished SARS-CoV-2 infection from influenza A (H1N1) infection and healthy controls with 95% accuracy. Distinct miRNA profiles were also observed in COVID-19 patients requiring oxygenation. This study demonstrates that SARS-CoV-2 infection induces a robust host miRNA response that could improve COVID-19 detection and patient management.

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.

Machine Learning Identifies Cellular and Exosomal MicroRNA Signatures of Lyssavirus Infection in Human Stem Cell-Derived Neurons.

Despite being vaccine preventable, rabies (lyssavirus) still has a significant impact on global mortality, disproportionally affecting children under 15 years of age. This neurotropic virus is deft at avoiding the immune system while travelling through neurons to the brain. Until recently, research efforts into the role of non-coding RNAs in rabies pathogenicity and detection have been hampered by a lack of human in vitro neuronal models. Here, we utilized our previously described human stem cell-derived neural model to investigate the effect of lyssavirus infection on microRNA (miRNA) expression in human neural cells and their secreted exosomes. Conventional differential expression analysis identified 25 cellular and 16 exosomal miRNAs that were significantly altered (FDR adjusted P-value <0.05) in response to different lyssavirus strains. Supervised machine learning algorithms determined 6 cellular miRNAs (miR-99b-5p, miR-346, miR-5701, miR-138-2-3p, miR-651-5p, and miR-7977) were indicative of lyssavirus infection (100% accuracy), with the first four miRNAs having previously established roles in neuronal function, or panic and impulsivity-related behaviors. Another 4-miRNA signatures in exosomes (miR-25-3p, miR-26b-5p, miR-218-5p, miR-598-3p) can independently predict lyssavirus infected cells with >99% accuracy. Identification of these robust lyssavirus miRNA signatures offers further insight into neural lineage responses to infection and provides a foundation for utilizing exosome miRNAs in the development of next-generation molecular diagnostics for rabies.

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.

Probe capture enrichment next-generation sequencing of complete foot-and-mouth disease virus genomes in clinical samples.

Next-generation sequencing (NGS) techniques offer an unprecedented "step-change" increase in the quantity and quality of sequence data rapidly generated from a sample and can be applied to obtain ultra-deep coverage of viral genomes. This is not possible with the routinely used Sanger sequencing method that gives the consensus reads, or by cloning approaches. In this study, a targeted-enrichment methodology for the simultaneous acquisition of complete foot-and-mouth disease virus (FMDV) genomes directly from clinical samples is presented. Biotinylated oligonucleotide probes (120 nt) were used to capture and enrich viral RNA following library preparation. To create a virus capture panel targeting serotype O and A simultaneously, 18 baits targeting the highly conserved regions of the 8.3 kb FMDV genome were synthesised, with 14 common to both serotypes, 2 specific to serotype O and 2 specific to serotype A. These baits were used to capture and enrich FMDV RNA (as cDNA) from samples collected during one pathogenesis and two vaccine efficacy trials, where pigs were infected with serotype O or A viruses. After enrichment, FMDV-specific sequencing reads increased by almost 3000-fold. The sequence data were used in variant call analysis to identify single nucleotide polymorphisms (SNPs). This methodology was robust in its ability to capture diverse sequences, was shown to be highly sensitive, and can be easily scaled for large-scale epidemiological studies.

Deep sequencing of primary human lung epithelial cells challenged with H5N1 influenza virus reveals a proviral role for CEACAM1.

Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 (H1N1) virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies.

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.

Dual microRNA Screens Reveal That the Immune-Responsive miR-181 Promotes Henipavirus Entry and Cell-Cell Fusion.

Hendra and Nipah viruses (family Paramyxoviridae, genus Henipavirus) are bat-borne viruses that cause fatal disease in humans and a range of other mammalian species. Gaining a deeper understanding of host pathways exploited by henipaviruses for infection may identify targets for new anti-viral therapies. Here we have performed genome-wide high-throughput agonist and antagonist screens at biosafety level 4 to identify host-encoded microRNAs (miRNAs) impacting henipavirus infection in human cells. Members of the miR-181 and miR-17~93 families strongly promoted Hendra virus infection. miR-181 also promoted Nipah virus infection, but did not affect infection by paramyxoviruses from other genera, indicating specificity in the virus-host interaction. Infection promotion was primarily mediated via the ability of miR-181 to significantly enhance henipavirus-induced membrane fusion. Cell signalling receptors of ephrins, namely EphA5 and EphA7, were identified as novel negative regulators of henipavirus fusion. The expression of these receptors, as well as EphB4, were suppressed by miR-181 overexpression, suggesting that simultaneous inhibition of several Ephs by the miRNA contributes to enhanced infection and fusion. Immune-responsive miR-181 levels was also up-regulated in the biofluids of ferrets and horses infected with Hendra virus, suggesting that the host innate immune response may promote henipavirus spread and exacerbate disease severity. This study is the first genome-wide screen of miRNAs influencing infection by a clinically significant mononegavirus and nominates select miRNAs as targets for future anti-viral therapy development.

Contraction of the type I IFN locus and unusual constitutive expression of IFN-α in bats.

Bats harbor many emerging and reemerging viruses, several of which are highly pathogenic in other mammals but cause no clinical signs of disease in bats. To determine the role of interferons (IFNs) in the ability of bats to coexist with viruses, we sequenced the type I IFN locus of the Australian black flying fox, Pteropus alecto, providing what is, to our knowledge, the first gene map of the IFN region of any bat species. Our results reveal a highly contracted type I IFN family consisting of only 10 IFNs, including three functional IFN-α loci. Furthermore, the three IFN-α genes are constitutively expressed in unstimulated bat tissues and cells and their expression is unaffected by viral infection. Constitutively expressed IFN-α results in the induction of a subset of IFN-stimulated genes associated with antiviral activity and resistance to DNA damage, providing evidence for a unique IFN system that may be linked to the ability of bats to coexist with viruses.

Development of multiplexed bead arrays for the simultaneous detection of nucleic acid from multiple viruses in bat samples.

Virus surveillance of wildlife populations is important for identifying, monitoring, and predicting the emergence of pathogens that pose a potential threat to animal and human health. Bats are identified as important wildlife hosts of many viruses capable of causing fatal human disease, including members of the henipaviruses, coronaviruses, rhabdoviruses and filoviruses. As global warming and habitat change are thought to impact upon pathogen transmission dynamics and increase the risk of spillover, virus surveillance in bat populations remains a significant component of efforts to improve the prediction and control of potential future disease outbreaks caused by bat-borne viruses. In this study we have developed two fluid bead array assays containing customized panels that target multiple bat-borne viruses. These assays detect up to 11 viral RNA's simultaneously in urine samples collected from wild bat populations in Australia and Bangladesh. The assays developed show high specificity for the target viruses and the analytical sensitivity compares favorably to qRT-PCR. These assays enhance the ability to monitor multi-pathogen dynamics and identify patterns of virus shedding from bat populations, thus informing key approaches to outbreak response and control.

Characterisation of novel microRNAs in the Black flying fox (Pteropus alecto) by deep sequencing.

BACKGROUND: Bats are a major source of new and emerging viral diseases. Despite the fact that bats carry and shed highly pathogenic viruses including Ebola, Nipah and SARS, they rarely display clinical symptoms of infection. Host factors influencing viral replication are poorly understood in bats and are likely to include both pre- and post-transcriptional regulatory mechanisms. MicroRNAs are a major mechanism of post-transcriptional gene regulation, however very little is known about them in bats. RESULTS: This study describes 399 microRNAs identified by deep sequencing of small RNA isolated from tissues of the Black flying fox, Pteropus alecto, a confirmed natural reservoir of the human pathogens Hendra virus and Australian bat lyssavirus. Of the microRNAs identified, more than 100 are unique amongst vertebrates, including a subset containing mutations in critical seed regions. Clusters of rapidly-evolving microRNAs were identified, as well as microRNAs predicted to target genes involved in antiviral immunity, the DNA damage response, apoptosis and autophagy. Closer inspection of the predicted targets for several highly supported novel miRNA candidates suggests putative roles in host-virus interaction. CONCLUSIONS: MicroRNAs are likely to play major roles in regulating virus-host interaction in bats, via dampening of inflammatory responses (limiting the effects of immunopathology), and directly limiting the extent of viral replication, either through restricting the availability of essential factors or by controlling apoptosis. Characterisation of the bat microRNA repertoire is an essential step towards understanding transcriptional regulation during viral infection, and will assist in the identification of mechanisms that enable bats to act as natural virus reservoirs. This in turn will facilitate the development of antiviral strategies for use in humans and other species.

Studying immunity to zoonotic diseases in the natural host - keeping it real.

Zoonotic viruses that emerge from wildlife and domesticated animals pose a serious threat to human and animal health. In many instances, mouse models have improved our understanding of the human immune response to infection; however, when dealing with emerging zoonotic diseases, they may be of limited use. This is particularly the case when the model fails to reproduce the disease status that is seen in the natural reservoir, transmission species or human host. In this Review, we discuss how researchers are placing more emphasis on the study of the immune response to zoonotic infections in the natural reservoir hosts and spillover species. Such studies will not only lead to a greater understanding of how these infections induce variable disease and immune responses in distinct species but also offer important insights into the evolution of mammalian immune systems.

Bat Mx1 and Oas1, but not Pkr are highly induced by bat interferon and viral infection.

Bats harbour many emerging and re-emerging viruses, several of which are highly pathogenic in other mammals but cause no diseases in bats. As the interferon (IFN) response represents a first line of defence against viral infection, the ability of bats to control viral replication may be linked to the activation of the IFN system. The three most studied antiviral IFN-stimulated genes (ISGs) in other mammals; Pkr, Mx1 and Oas1 were examined in our model bat species, Pteropus alecto. Our results demonstrate that the three ISGs from P. alecto are highly conserved in their functional domains and promoter elements compared to corresponding genes from other mammals. However, P. alecto Oas1 contains two IFN-stimulated response elements (ISRE) in its promoter region compared with the single ISRE present in human OAS1 which may lead to higher IFN inducibility of the bat gene. Both Oas1 and Mx1 were induced in a highly IFN-dependent manner following stimulation with IFN or synthetic double-strand RNA (dsRNA) whereas Pkr showed evidence of being induced in an IFN-independent manner. Furthermore, bat Oas1 appeared to be the most inducible of the three ISGs following either IFN stimulation or viral infection, providing evidence that Oas1 may play a more important role in antiviral activity in bats compared with Mx1 or Pkr. Our results have important implications for the different roles of ISGs in bats and provide the first step in understanding the role of these molecules in the ability of bats to coexist with viruses.

Purification and characterisation of immunoglobulins from the Australian black flying fox (Pteropus alecto) using anti-fab affinity chromatography reveals the low abundance of IgA.

There is now an overwhelming body of evidence that implicates bats in the dissemination of a long list of emerging and re-emerging viral agents, often causing illnesses or death in both animals and humans. Despite this, there is a paucity of information regarding the immunological mechanisms by which bats coexist with highly pathogenic viruses. Immunoglobulins are major components of the adaptive immune system. Early studies found bats may have quantitatively lower antibody responses to model antigens compared to conventional laboratory animals. To further understand the antibody response of bats, the present study purified and characterised the major immunoglobulin classes from healthy black flying foxes, Pteropus alecto. We employed a novel strategy, where IgG was initially purified and used to generate anti-Fab specific antibodies. Immobilised anti-Fab specific antibodies were then used to capture other immunoglobulins from IgG depleted serum. While high quantities of IgM were successfully isolated from serum, IgA was not. Only trace quantities of IgA were detected in the serum by mass spectrometry. Immobilised ligands specific to IgA (Jacalin, Peptide M and staphylococcal superantigen-like protein) also failed to capture P. alecto IgA from serum. IgM was the second most abundant serum antibody after IgG. A survey of mucosal secretions found IgG was the dominant antibody class rather than IgA. Our study demonstrates healthy P. alecto bats have markedly less serum IgA than expected. Higher quantities of IgG in mucosal secretions may be compensation for this low abundance or lack of IgA. Knowledge and reagents developed within this study can be used in the future to examine class-specific antibody response within this important viral host.

Genetic architecture of gene expression in the chicken.

BACKGROUND: The annotation of many genomes is limited, with a large proportion of identified genes lacking functional assignments. The construction of gene co-expression networks is a powerful approach that presents a way of integrating information from diverse gene expression datasets into a unified analysis which allows inferences to be drawn about the role of previously uncharacterised genes. Using this approach, we generated a condition-free gene co-expression network for the chicken using data from 1,043 publically available Affymetrix GeneChip Chicken Genome Arrays. This data was generated from a diverse range of experiments, including different tissues and experimental conditions. Our aim was to identify gene co-expression modules and generate a tool to facilitate exploration of the functional chicken genome. RESULTS: Fifteen modules, containing between 24 and 473 genes, were identified in the condition-free network. Most of the modules showed strong functional enrichment for particular Gene Ontology categories. However, a few showed no enrichment. Transcription factor binding site enrichment was also noted. CONCLUSIONS: We have demonstrated that this chicken gene co-expression network is a useful tool in gene function prediction and the identification of putative novel transcription factors and binding sites. This work highlights the relevance of this methodology for functional prediction in poorly annotated genomes such as the chicken.

Comparative analysis of bat genomes provides insight into the evolution of flight and immunity.

Bats are the only mammals capable of sustained flight and are notorious reservoir hosts for some of the world's most highly pathogenic viruses, including Nipah, Hendra, Ebola, and severe acute respiratory syndrome (SARS). To identify genetic changes associated with the development of bat-specific traits, we performed whole-genome sequencing and comparative analyses of two distantly related species, fruit bat Pteropus alecto and insectivorous bat Myotis davidii. We discovered an unexpected concentration of positively selected genes in the DNA damage checkpoint and nuclear factor κB pathways that may be related to the origin of flight, as well as expansion and contraction of important gene families. Comparison of bat genomes with other mammalian species has provided new insights into bat biology and evolution.