The host transcriptional response to superinfection by influenza A virus and Streptococcus pneumoniae.

Secondary bacterial challenges during influenza virus infection "superinfection") cause excessive mortality and hospitalization. Here, we present a longitudinal study of bulk gene expression changes in murine lungs during superinfection, with an initial influenza A virus infection and a subsequent Streptococcus pneumoniae infection. In addition to the well-characterized impairment of the host response, we identified superinfection-specific alterations in the global transcriptional program that are linked to the host's ability to resist the pathogens. Particularly, whereas superinfected mice manifested an excessive rapid induction of the resistance-to-infection program, there was a substantial tissue-level rewiring of this program: upon superinfection, interferon-regulated genes were switched from positive to negative correlations with the host's resistance state, whereas genes of fatty acid metabolism switched from negative to positive correlations with resistance states. Thus, the transcriptional resistance state in superinfection is reprogrammed toward repressed interferon signaling and induced fatty acid metabolism. Our findings suggest new insights into a tissue-level remodeling of the host defense upon superinfection, providing promising targets for future therapeutic interventions. IMPORTANCE: Secondary bacterial infections are the most frequent complications during influenza A virus (IAV) pandemic outbreaks, contributing to excessive morbidity and mortality in the human population. Most IAV-related deaths are attributed to Streptococcus pneumoniae (SP) infections, which usually begin within the first week of IAV infection in the respiratory tracts. Here, we focused on longitudinal transcriptional responses during a superinfection model consisting of an SP infection that follows an initial IAV infection, comparing superinfection to an IAV-only infection, an SP-only infection, and control treatments. Our longitudinal data allowed a fine analysis of gene expression changes during superinfection. For instance, we found that superinfected mice exhibited rapid gene expression induction or reduction within the first 12 h after encountering the second pathogen. Cell proliferation and immune response activation processes were upregulated, while endothelial processes, vasculogenesis, and angiogenesis were downregulated, providing promising targets for future therapeutic interventions. We further analyzed the longitudinal transcriptional responses in the context of a previously defined spectrum of the host's resistance state, revealing superinfection-specific reprogramming of resistance states, such as reprogramming of fatty acid metabolism and interferon signaling. The reprogrammed functions are compelling new targets for switching the pathogenic superinfection state into a single-infection state.

Lipocalin-2 regulates the expression of interferon-stimulated genes and the susceptibility of prostate cancer cells to oncolytic virus infection.

Lipocalin-2 (LCN2) performs pleiotropic and tumor context-dependent functions in cancers of diverse etiologies. In prostate cancer (PCa) cells, LCN2 regulates distinct phenotypic features, including cytoskeleton organization and expression of inflammation mediators. Oncolytic virotherapy uses oncolytic viruses (OVs) to kill cancer cells and induce anti-tumor immunity. A main source of specificity of OVs towards tumor cells stems from cancer-induced defects in interferon (IFN)-based cell autonomous immune responses. However, the molecular underpinnings of such defects in PCa cells are only partially understood. Moreover, LCN2 effects on IFN responses of PCa cells and their susceptibility to OVs are unknown. To examine these issues, we queried gene expression databases for genes coexpressed with LCN2, revealing co-expression of IFN-stimulated genes (ISGs) and LCN2. Analysis of human PCa cells revealed correlated expression of LCN2 and subsets of IFNs and ISGs. CRISPR/Cas9-mediated stable knockout of LCN2 in PC3 cells or transient overexpression of LCN2 in LNCaP cells revealed LCN2-mediated regulation of IFNE (and IFNL1) expression, activation of JAK/STAT pathway, and expression of selected ISGs. Accordingly, and dependent on a functional JAK/STAT pathway, LCN2 reduced the susceptibility of PCa cells to infection with the IFN-sensitive OV, EHDV-TAU. In PC3 cells, LCN2 knockout increased phosphorylation of eukaryotic initiation factor 2α (p-eIF2α). Inhibition of PKR-like ER kinase (PERK) in PC3-LCN2-KO cells reduced p-eIF2α while increasing constitutive IFNE expression, phosphorylation of STAT1, and ISG expression; and decreasing EHDV-TAU infection. Together, these data propose that LCN2 regulates PCa susceptibility to OVs through attenuation of PERK activity and increased IFN and ISG expression.

Discovery of an unrecognized nidovirus associated with granulomatous hepatitis in rainbow trout.

Rainbow trout (Oncorhynchus mykiss) is the principal species of inland-farmed fish in the Western hemisphere. Recently, we diagnosed in farmed rainbow trout a disease in which the hallmark is granulomatous-like hepatitis. No biotic agents could be isolated from lesions. Still, unbiased high-throughput sequencing and bioinformatics analyses revealed the presence of a novel piscine nidovirus that we named "Trout Granulomatous Virus" (TGV). TGV genome (28,767 nucleotides long) is predicted to encode non-structural (1a and 1 ab) and structural (S, M, and N) proteins that resemble proteins of other known piscine nidoviruses. High loads of TGV transcripts were detected by quantitative RT-PCR in diseased fish and visualized in hepatic granulomatous sites by fluorescence in situ hybridization. Transmission electron microscopy (TEM) revealed coronavirus-like particles in these lesions. Together, these analyses corroborated the association of TGV with the lesions. The identification and detection of TGV provide means to control TGV spread in trout populations.

Distinct gene programs underpinning disease tolerance and resistance in influenza virus infection.

When challenged with an invading pathogen, the host-defense response is engaged to eliminate the pathogen (resistance) and to maintain health in the presence of the pathogen (disease tolerance). However, the identification of distinct molecular programs underpinning disease tolerance and resistance remained obscure. We exploited transcriptional and physiological monitoring across 33 mouse strains, during in vivo influenza virus infection, to identify two host-defense gene programs-one is associated with hallmarks of disease tolerance and the other with hallmarks of resistance. Both programs constitute generic responses in multiple mouse and human cell types. Our study describes the organizational principles of these programs and validates Arhgdia as a regulator of disease-tolerance states in epithelial cells. We further reveal that the baseline disease-tolerance state in peritoneal macrophages is associated with the pathophysiological response to injury and infection. Our framework provides a paradigm for the understanding of disease tolerance and resistance at the molecular level.

Ultrasensitive chemiluminescent neuraminidase probe for rapid screening and identification of small-molecules with antiviral activity against influenza A virus in mammalian cells.

Influenza A virus is the most virulent influenza subtype and is associated with large-scale global pandemics characterized by high levels of morbidity and mortality. Developing simple and sensitive molecular methods for detecting influenza viruses is critical. Neuraminidase, an exo-glycosidase displayed on the surface of influenza virions, is responsible for the release of the virions and their spread in the infected host. Here, we present a new phenoxy-dioxetane chemiluminescent probe (CLNA) that can directly detect neuraminidase activity. The probe exhibits an effective turn-on response upon reaction with neuraminidase and produces a strong emission signal at 515 nm with an extremely high signal-to-noise ratio. Comparison measurements of our new probe with previously reported analogous neuraminidase optical probes showed superior detection capability in terms of response time and sensitivity. Thus, as far as we know, our probe is the most sensitive neuraminidase probe known to date. The chemiluminescence turn-on response produced by our neuraminidase probe enables rapid screening for small molecules that inhibit viral replication through different mechanisms as validated directly in influenza A-infected mammalian cells using the known inhibitors oseltamivir and amantadine. We expect that our new chemiluminescent neuraminidase probe will prove useful for various applications requiring neuraminidase detection including drug discovery assays against various influenza virus strains in mammalian cells.

Inhibition of Respiratory RNA Viruses by a Composition of Ionophoric Polyphenols with Metal Ions.

Controlling the infectivity of respiratory RNA viruses is critical, especially during the current SARS-CoV-2 pandemic. There is an unmet need for therapeutic agents that can reduce viral replication, preferably independent of the accumulation of viral mutations. Zinc ions have an apparent activity as modulators of intracellular viral RNA replication and thus, appear attractive in reducing viral RNA load and infectivity. However, the intracellular concentration of zinc is usually too low for achieving an optimal inhibitory effect. Various herbal polyphenols serve as excellent zinc ionophores with known antiviral properties. Here, we combined zinc picolinate with a collection of flavonoids, representing commonly used polyphenols. Copper was added to avoid ionic imbalance during treatment and to improve efficacy. Each component separately, as well as their combinations, did not interfere with the viability of cultured A549, H1299, or Vero cells in vitro as determined by MTT assay. The safe combinations were further evaluated to determine antiviral activity. Fluorescence-activated cell sorting and quantitative polymerase chain reaction were used to evaluate antiviral activity of the combinations. They revealed a remarkable (50-95%) decrease, in genome replication levels of a diverse group of respiratory RNA viruses, including the human coronavirus OC43 (HCoV-OC43; a betacoronavirus that causes the common cold), influenza A virus (IAV, strain A/Puerto Rico/8/34 H1N1), and human metapneumovirus (hMPV). Collectively, our results offer an orally bioavailable therapeutic approach that is non-toxic, naturally sourced, applicable to numerous RNA viruses, and potentially insensitive to new mutations and variants.

Inferring Protein Function in an Emerging Virus: Detection of the Nucleoprotein in Tilapia Lake Virus.

Emerging viruses impose global threats to animal and human populations and may bear novel genes with limited homology to known sequences, necessitating the development of novel approaches to infer and test protein functions. This challenge is dramatically evident in tilapia lake virus (TiLV), an emerging "orthomyxo-like" virus that threatens the global tilapia aquaculture and food security of millions of people. The majority of TiLV proteins have no homology to known sequences, impeding functionality assessments. Using a novel bioinformatics approach, we predicted that TiLV's Protein 4 encodes the nucleoprotein, a factor essential for viral RNA replication. Multiple methodologies revealed the expected properties of orthomyxoviral nucleoproteins. A modified yeast three-hybrid assay detected Protein 4-RNA interactions, which were independent of the RNA sequence, and identified specific positively charged residues involved. Protein 4-RNA interactions were uncovered by R-DeeP and XRNAX methodologies. Immunoelectron microscopy found that multiple Protein 4 copies localized along enriched ribonucleoproteins. TiLV RNA from cells and virions coimmunoprecipitated with Protein 4. Immunofluorescence microscopy detected Protein 4 in the cytoplasm and nuclei, and nuclear Protein 4 increased upon CRM1 inhibition, suggesting CRM1-dependent nuclear export of TiLV RNA. Together, these data reveal TiLV's nucleoprotein and highlight the ability to infer protein functionality, including novel RNA-binding proteins, in emerging pathogens. These are important in light of the expected discovery of many unknown viruses and the zoonotic potential of such pathogens. IMPORTANCE Tilapia is an important source of dietary protein, especially in developing countries. Massive losses of tilapia were identified worldwide, risking the food security of millions of people. Tilapia lake virus (TiLV) is an emerging pathogen responsible for these disease outbreaks. TiLV's genome encodes 10 major proteins, 9 of which show no homology to other known viral or cellular proteins, hindering functionality assessment of these proteins. Here, we describe a novel bioinformatics approach to infer the functionality of TiLV proteins, which predicted Protein 4 as the nucleoprotein, a factor essential for viral RNA replication. We provided experimental support for this prediction by applying multiple molecular, biochemical, and imaging approaches. Overall, we illustrate a strategy for functional analyses in viral discovery. The strategy is important in light of the expected discovery of many unknown viruses and the zoonotic potential of such pathogens.

Mapping of Tilapia Lake Virus entry pathways with inhibitors reveals dependence on dynamin activity and cholesterol but not endosomal acidification.

Tilapia Lake Virus (TiLV) is an emerging virus lethal to tilapia, which threatens the global tilapia aquaculture with severe implications for food security. TiLV possesses similar features to orthomyxoviruses but is classified in the sole and the monotypic genus Tilapinevirus of the family Amnoonviridae. TiLV enveloped virions encapsidate a genome comprising ten segments of single-stranded, negative RNA. Remarkably, nine of TiLV's ten major proteins lack sequence homology to any known viral or cellular proteins. The mode of TiLV entry into tilapia cells is not known. Following the measurement of the entry window of TiLV (∼3 h), we applied a panel of inhibitors of known regulators of endocytic functions to map the molecular requirements for TiLV entry. We identified productive entry by quantification of TiLV nucleoprotein expression and the generation of infectious particles. Inhibition of dynamin activity with dynasore or dynole, or depletion of cholesterol with methyl-ß-cyclodextrin, strongly inhibited TiLV protein synthesis and infectious virion production. Moreover, inhibition of actin cytoskeleton polymerization with latrunculin A or microtubule polymerization with nocodazole within the entry window resulted in partial inhibition of TiLV infection. In contrast, inhibitors of endosomal acidification (NH4Cl, bafilomycin A1, or chloroquine), an inhibitor of clathrin-coated pit assembly (pitstop 2), and erlotinib-an inhibitor of the endocytic Cyclin G-associated kinase (GAK), did not affect TiLV entry. Altogether, these results suggest that TiLV enters via dynamin-mediated endocytosis in a cholesterol-, cytoskeleton-dependent manner, and clathrin-, pH-independent manner. Thus, despite being an orthomyxo-like virus, when compared to the prototypical orthomyxovirus (influenza A virus), TiLV shows a distinct set of requirements for entry into cells.

Oncolytic Virotherapy: The Cancer Cell Side.

Cell autonomous immunity genes mediate the multiple stages of anti-viral defenses, including recognition of invading pathogens, inhibition of viral replication, reprogramming of cellular metabolism, programmed-cell-death, paracrine induction of antiviral state, and activation of immunostimulatory inflammation. In tumor development and/or immunotherapy settings, selective pressure applied by the immune system results in tumor immunoediting, a reduction in the immunostimulatory potential of the cancer cell. This editing process comprises the reduced expression and/or function of cell autonomous immunity genes, allowing for immune-evasion of the tumor while concomitantly attenuating anti-viral defenses. Combined with the oncogene-enhanced anabolic nature of cancer-cell metabolism, this attenuation of antiviral defenses contributes to viral replication and to the selectivity of oncolytic viruses (OVs) towards malignant cells. Here, we review the manners by which oncogene-mediated transformation and tumor immunoediting combine to alter the intracellular milieu of tumor cells, for the benefit of OV replication. We also explore the functional connection between oncogenic signaling and epigenetic silencing, and the way by which restriction of such silencing results in immune activation. Together, the picture that emerges is one in which OVs and epigenetic modifiers are part of a growing therapeutic toolbox that employs activation of anti-tumor immunity for cancer therapy.

Leveraging the cell lineage to predict cell-type specificity of regulatory variation from bulk genomics.

Recent computational methods have enabled the inference of the cell-type-specificity of eQTLs based on bulk transcriptomes from highly heterogeneous tissues. However, these methods are limited in their scalability to highly heterogeneous tissues and limited in their broad applicability to any cell-type specificity of eQTLs. Here we present and demonstrate Cell Lineage Genetics (CeL-Gen), a novel computational approach that allows inference of eQTLs together with the subsets of cell types in which they have an effect, from bulk transcriptome data. To obtain improved scalability and broader applicability, CeL-Gen takes as input the known cell lineage tree and relies on the observation that dynamic changes in genetic effects occur relatively infrequently during cell differentiation. CeL-Gen can therefore be used not only to tease apart genetic effects derived from different cell types but also to infer the particular differentiation steps in which genetic effects are altered.

Modeling SARS-CoV-2 Infection in Mice Using Lentiviral hACE2 Vectors Infers Two Modes of Immune Responses to SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a severe global pandemic. Mice models are essential to investigate infection pathology, antiviral drugs, and vaccine development. However, wild-type mice lack the human angiotensin-converting enzyme 2 (hACE2) that mediates SARS-CoV-2 entry into human cells and consequently are not susceptible to SARS-CoV-2 infection. hACE2 transgenic mice could provide an efficient COVID-19 model, but are not always readily available, and practically restricted to specific strains. Therefore, there is a dearth of additional mouse models for SARS-CoV-2 infection. We applied lentiviral vectors to generate hACE2 expression in interferon receptor knock-out (IFNAR1-/-) mice. Lenti-hACE2 transduction supported SARS-CoV-2 replication in vivo, simulating mild acute lung disease. Gene expression analysis revealed two modes of immune responses to SARS-CoV-2 infection: one in response to the exposure of mouse lungs to SARS-CoV-2 particles in the absence of productive viral replication, and the second in response to productive SARS-CoV-2 infection. Our results infer that immune response to immunogenic elements on incoming virus or in productively infected cells stimulate diverse immune effectors, even in absence of type I IFN signaling. Our findings should contribute to a better understanding of the immune response triggered by SARS-CoV-2 and to further elucidate COVID-19.

Constitutive low expression of antiviral effectors sensitizes melanoma cells to a novel oncolytic virus.

STAT1 is a critical effector and a target gene of interferon (IFN) signaling, and thus a central mediator of antiviral responses. As both a mediator and a target of IFN signals, STAT1 expression reports on, and determines IFN activity. Gene expression analyses of melanoma patient samples revealed varied levels of STAT1 expression, which highly correlated with expression of >700 genes. The ability of oncolytic viruses to exploit tumor-induced defects to antiviral responses suggests that oncolytic viruses may efficiently target a subset of melanomas, yet these should be defined. We modeled this scenario with murine B16F10 melanomas, immortalized skin fibroblasts as controls and a novel oncolytic virus, EHDV-TAU. In B16F10 cells, constitutive low expression of STAT1 and its target genes, which included intracellular pattern recognition receptors (PRRs), correlated with their inability to mount IFN-based antiviral responses upon EHDV-TAU challenge, and with potency of EHDV-TAU-induced oncolysis. This underexpression of interferon stimulated genes (ISGs) and PRRs, and the inability of EHDV-TAU to induce their expression, were reversed by epigenetic modifiers, suggesting epigenetic silencing as a basis for their underexpression. Despite their inability to mount IFN/STAT-based responses upon viral infection, EHDV-TAU infected B16F10 cells secreted immune-stimulatory chemokines. Accordingly, in vivo, EHDV-TAU enhanced intratumoral infiltration of cytotoxic T-cells and reduced growth of local and distant tumors. We propose that "STAT1 signatures" should guide melanoma virotherapy treatments, and that oncolytic viruses such as EHDV-TAU have the potential to exploit the cellular context of low-STAT1 tumors.

Human Metapneumovirus Escapes NK Cell Recognition through the Downregulation of Stress-Induced Ligands for NKG2D.

The Pneumoviridae family includes human metapneumovirus (HMPV) and human orthopneumovirus, which is also known as a respiratory syncytial virus (HRSV). These are large enveloped, negative single-strand RNA viruses. HMPV and HRSV are the human members, which commonly infect children. HMPV, which was discovered in 2001, infects most children until the age of five, which causes an influenza-like illness. The interaction of this virus with immune cells is poorly understood. In this study, we show that HMPV evades natural killer (NK) cell attack by downregulating stress-induced ligands for the activating receptor NKG2D including: Major histocompatibility complex (MHC) class I polypeptide-related sequences A and B (MICA, MICB), UL16 binding proteins ULBP2, and ULBP3, but not ULBP1. Mechanistically, we show that the viral protein G is involved in the downregulation of ULBP2 and that the viral protein M2.2 is required for MICA and MICB downregulation. These findings emphasize the importance of NK cells, in general, and NKG2D, in particular, in controlling HMPV infection, which opens new avenues for treating HMPV.

Predicting Phenotypic Diversity from Molecular and Genetic Data.

Despite the importance of complex phenotypes, an in-depth understanding of the combined molecular and genetic effects on a phenotype has yet to be achieved. Here, we introduce InPhenotype, a novel computational approach for complex phenotype prediction, where gene-expression data and genotyping data are integrated to yield quantitative predictions of complex physiological traits. Unlike existing computational methods, InPhenotype makes it possible to model potential regulatory interactions between gene expression and genomic loci without compromising the continuous nature of the molecular data. We applied InPhenotype to synthetic data, exemplifying its utility for different data parameters, as well as its superiority compared to current methods in both prediction quality and the ability to detect regulatory interactions of genes and genomic loci. Finally, we show that InPhenotype can provide biological insights into both mouse and yeast datasets.

Cell composition analysis of bulk genomics using single-cell data.

Single-cell RNA sequencing (scRNA-seq) is a rich resource of cellular heterogeneity, opening new avenues in the study of complex tissues. We introduce Cell Population Mapping (CPM), a deconvolution algorithm in which reference scRNA-seq profiles are leveraged to infer the composition of cell types and states from bulk transcriptome data ('scBio' CRAN R-package). Analysis of individual variations in lungs of influenza-virus-infected mice reveals that the relationship between cell abundance and clinical symptoms is a cell-state-specific property that varies gradually along the continuum of cell-activation states. The gradual change is confirmed in subsequent experiments and is further explained by a mathematical model in which clinical outcomes relate to cell-state dynamics along the activation process. Our results demonstrate the power of CPM in reconstructing the continuous spectrum of cell states within heterogeneous tissues.

Dissection of Influenza Infection In Vivo by Single-Cell RNA Sequencing.

The influenza virus is a major cause of morbidity and mortality worldwide. Yet, both the impact of intracellular viral replication and the variation in host response across different cell types remain uncharacterized. Here we used single-cell RNA sequencing to investigate the heterogeneity in the response of lung tissue cells to in vivo influenza infection. Analysis of viral and host transcriptomes in the same single cell enabled us to resolve the cellular heterogeneity of bystander (exposed but uninfected) as compared with infected cells. We reveal that all major immune and non-immune cell types manifest substantial fractions of infected cells, albeit at low viral transcriptome loads relative to epithelial cells. We show that all cell types respond primarily with a robust generic transcriptional response, and we demonstrate novel markers specific for influenza-infected as opposed to bystander cells. These findings open new avenues for targeted therapy aimed exclusively at infected cells.

Interleukin-6 and Interferon-α Signaling via JAK1-STAT Differentially Regulate Oncolytic versus Cytoprotective Antiviral States.

Malignancy-induced alterations to cytokine signaling in tumor cells differentially regulate their interactions with the immune system and oncolytic viruses. The abundance of inflammatory cytokines in the tumor microenvironment suggests that such signaling plays key roles in tumor development and therapy efficacy. The JAK-STAT axis transduces signals of interleukin-6 (IL-6) and interferons (IFNs), mediates antiviral responses, and is frequently altered in prostate cancer (PCa) cells. However, how activation of JAK-STAT signaling with different cytokines regulates interactions between oncolytic viruses and PCa cells is not known. Here, we employ LNCaP PCa cells, expressing (or not) JAK1, activated (or not) with IFNs (α or γ) or IL-6, and infected with RNA viruses of different oncolytic potential (EHDV-TAU, hMPV-GFP, or HIV-GFP) to address this matter. We show that in JAK1-expressing cells, IL-6 sensitized PCa cells to viral cell death in the presence or absence of productive infection, with dependence on virus employed. Contrastingly, IFNα induced a cytoprotective antiviral state. Biochemical and genetic (knockout) analyses revealed dependency of antiviral state or cytoprotection on STAT1 or STAT2 activation, respectively. In IL-6-treated cells, STAT3 expression was required for anti-proliferative signaling. Quantitative proteomics (SILAC) revealed a core repertoire of antiviral IFN-stimulated genes, induced by IL-6 or IFNs. Oncolysis in the absence of productive infection, induced by IL-6, correlated with reduction in regulators of cell cycle and metabolism. These results call for matching the viral features of the oncolytic agent, the malignancy-induced genetic-epigenetic alterations to JAK/STAT signaling and the cytokine composition of the tumor microenvironment for efficient oncolytic virotherapy.

NK-cell receptors NKp46 and NCR1 control human metapneumovirus infection.

Natural killer (NK) cells are capable of killing various pathogens upon stimulation of activating receptors. Human metapneumovirus (HMPV) is a respiratory virus, which was discovered in 2001 and is responsible for acute respiratory tract infection in infants and children worldwide. HMPV infection is very common, infecting around 70% of all children under the age of five. Under immune suppressive conditions, HMPV infection can be fatal. Not much is known on how NK cells respond to HMPV. In this study, using reporter assays and NK-cell cytotoxicity assays performed with human and mouse NK cells, we demonstrated that the NKp46-activating receptor and its mouse orthologue Ncr1, both members of the natural cytotoxicity receptor (NCR) family, recognized an unknown ligand expressed by HMPV-infected human cells. We demonstrated that MHC class I is upregulated and MICA is downregulated upon HMPV infection. We also characterized mouse NK-cell phenotype in the blood and the lungs of HMPV-infected mice and found that lung NK cells are more activated and expressing NKG2D, CD43, CD27, KLRG1, and CD69 compared to blood NK cells regardless of HMPV infection. Finally, we demonstrated, using Ncr1-deficient mice, that NCR1 plays a critical role in controlling HMPV infection.

Detection of Tilapia Lake Virus in Clinical Samples by Culturing and Nested Reverse Transcription-PCR.

Tilapia are an important group of farmed fish that serve as a significant protein source worldwide. In recent years, substantial mortality of wild tilapia has been observed in the Sea of Galilee and in commercial ponds in Israel and Ecuador. We have identified the etiological agent of these mass die-offs as a novel orthomyxo-like virus and named it tilapia lake virus (TiLV). Here, we provide the conditions for efficient isolation, culturing, and quantification of the virus, including the use of susceptible fish cell lines. Moreover, we describe a sensitive nested reverse transcription-PCR (RT-PCR) assay allowing the rapid detection of TiLV in fish organs. This assay revealed, for the first time to our knowledge, the presence of TiLV in diseased Colombian tilapia, indicating a wider distribution of this emerging pathogen and stressing the risk that TiLV poses for the global tilapia industry. Overall, the described procedures should provide the tilapia aquaculture industry with important tools for the detection and containment of this pathogen.

Suppression of human metapneumovirus (HMPV) infection by the innate sensing gene CEACAM1.

The innate sensing system is equipped with PRRs specialized in recognizing molecular structures (PAMPs) of various pathogens. This leads to the induction of anti-viral genes and inhibition of virus growth. Human Metapneumovirus (HMPV) is a major respiratory virus that causes an upper and lower respiratory tract infection in children. In this study we show that upon HMPV infection, the innate sensing system detects the viral RNA through the RIG-I sensor leading to induction of CEACAM1 expression. We further show that CEACAM1 is induced via binding of IRF3 to the CEACAM1 promoter. We demonstrate that induction of CEACAM1 suppresses the viral loads via inhibition of the translation machinery in the infected cells in an SHP2-dependent manner. In summary, we show here that HMPV-infected cells upregulates CEACAM1 to restrict HMPV infection.