Triclosan is a mitochondrial uncoupler in live zebrafish.

Triclosan (TCS) is a synthetic antimicrobial agent used in many consumer goods at millimolar concentrations. As a result of exposure, TCS has been detected widely in humans. We have recently discovered that TCS is a proton ionophore mitochondrial uncoupler in multiple types of living cells. Here, we present novel data indicating that TCS is also a mitochondrial uncoupler in a living organism: 24-hour post-fertilization (hpf) zebrafish embryos. These experiments were conducted using a Seahorse Bioscience XFe 96 Extracellular Flux Analyzer modified for bidirectional temperature control, using the XF96 spheroid plate to position and measure one zebrafish embryo per well. Using this method, after acute exposure to TCS, the basal oxygen consumption rate (OCR) increases, without a decrease in survival or heartbeat rate. TCS also decreases ATP-linked respiration and spare respiratory capacity and increases proton leak: all indicators of mitochondrial uncoupling. Our data indicate, that TCS is a mitochondrial uncoupler in vivo, which should be taken into consideration when assessing the toxicity and/or pharmaceutical uses of TCS. This is the first example of usage of a Seahorse Extracellular Flux Analyzer to measure bioenergetic flux of a single zebrafish embryo per well in a 96-well assay format. The method developed in this study provides a high-throughput tool to identify previously unknown mitochondrial uncouplers in a living organism. Copyright © 2016 John Wiley & Sons, Ltd.

Differential expression and ligand binding indicate alternative functions for zebrafish polymeric immunoglobulin receptor (pIgR) and a family of pIgR-like (PIGRL) proteins.

The polymeric immunoglobulin (Ig) receptor (pIgR) is an integral transmembrane glycoprotein that plays an important role in the mammalian immune response by transporting soluble polymeric Igs across mucosal epithelial cells. Single pIgR genes, which are expressed in lymphoid organs including mucosal tissues, have been identified in several teleost species. A single pigr gene has been identified on zebrafish chromosome 2 along with a large multigene family consisting of 29 pigr-like (PIGRL) genes. Full-length transcripts from ten different PIGRL genes that encode secreted and putative inhibitory membrane-bound receptors have been characterized. Although PIGRL and pigr transcripts are detected in immune tissues, only PIGRL transcripts can be detected in lymphoid and myeloid cells. In contrast to pIgR which binds Igs, certain PIGRL proteins bind phospholipids. PIGRL transcript levels are increased after infection with Streptococcus iniae, suggesting a role for PIGRL genes during bacterial challenge. Transcript levels of PIGRL genes are decreased after infection with Snakehead rhabdovirus, suggesting that viral infection may suppress PIGRL function.

Actin mediates the nanoscale membrane organization of the clustered membrane protein influenza hemagglutinin.

The influenza viral membrane protein hemagglutinin (HA) is required at high concentrations on virion and host-cell membranes for infectivity. Because the role of actin in membrane organization is not completely understood, we quantified the relationship between HA and host-cell actin at the nanoscale. Results obtained using superresolution fluorescence photoactivation localization microscopy (FPALM) in nonpolarized cells show that HA clusters colocalize with actin-rich membrane regions (ARMRs). Individual molecular trajectories in live cells indicate restricted HA mobility in ARMRs, and actin disruption caused specific changes to HA clustering. Surprisingly, the actin-binding protein cofilin was excluded from some regions within several hundred nanometers of HA clusters, suggesting that HA clusters or adjacent proteins within the same clusters influence local actin structure. Thus, with the use of imaging, we demonstrate a dynamic relationship between glycoprotein membrane organization and the actin cytoskeleton at the nanoscale.

Cell-specific mitotic defect and dyserythropoiesis associated with erythroid band 3 deficiency.

Most eukaryotic cell types use a common program to regulate the process of cell division. During mitosis, successful partitioning of the genetic material depends on spatially coordinated chromosome movement and cell cleavage. Here we characterize a zebrafish mutant, retsina (ret), that exhibits an erythroid-specific defect in cell division with marked dyserythropoiesis similar to human congenital dyserythropoietic anemia. Erythroblasts from ret fish show binuclearity and undergo apoptosis due to a failure in the completion of chromosome segregation and cytokinesis. Through positional cloning, we show that the ret mutation is in a gene (slc4a1) encoding the anion exchanger 1 (also called band 3 and AE1), an erythroid-specific cytoskeletal protein. We further show an association between deficiency in Slc4a1 and mitotic defects in the mouse. Rescue experiments in ret zebrafish embryos expressing transgenic slc4a1 with a variety of mutations show that the requirement for band 3 in normal erythroid mitosis is mediated through its protein 4.1R-binding domains. Our report establishes an evolutionarily conserved role for band 3 in erythroid-specific cell division and illustrates the concept of cell-specific adaptation for mitosis.

The gene history of zebrafish tlr4a and tlr4b is predictive of their divergent functions.

Mammalian immune responses to LPS exposure are typified by the robust induction of NF-kappaB and IFN-beta responses largely mediated by TLR4 signal transduction pathways. In contrast to mammals, Tlr4 signal transduction pathways in nontetrapods are not well understood. Comprehensive syntenic and phylogenetic analyses support our hypothesis that zebrafish tlr4a and tlr4b genes are paralogous rather than orthologous to human TLR4. Furthermore, we provide evidence to support our assertion that the in vivo responsiveness of zebrafish to LPS exposure is not mediated by Tlr4a and Tlr4b paralogs because they fail to respond to LPS stimulation in vitro. Zebrafish Tlr4a and Tlr4b paralogs were also unresponsive to heat-killed Escherichia coli and Legionella pneumophila. Using chimeric molecules in which portions of the zebrafish Tlr4 proteins were fused to portions of the mouse TLR4 protein, we show that the lack of responsiveness to LPS was most likely due to the inability of the extracellular portions of zebrafish Tlr4a and Tlr4b to recognize the molecule, rather than to changes in their capacities to transduce signals through their Toll/IL-1 receptor (TIR) domains. Taken together, these findings strongly support the notion that zebrafish tlr4a and tlr4b paralogs have evolved to provide alternative ligand specificities to the Tlr immune defense system in this species. These data demonstrate that intensive examination of gene histories when describing the Tlr proteins of basally diverging vertebrates is required to obtain fuller appreciation of the evolution of their function. These studies provide the first evidence for the functional evolution of a novel Tlr.

Modeling Virus-Induced Inflammation in Zebrafish: A Balance Between Infection Control and Excessive Inflammation.

The inflammatory response to viral infection in humans is a dynamic process with complex cell interactions that are governed by the immune system and influenced by both host and viral factors. Due to this complexity, the relative contributions of the virus and host factors are best studied in vivo using animal models. In this review, we describe how the zebrafish (Danio rerio) has been used as a powerful model to study host-virus interactions and inflammation by combining robust forward and reverse genetic tools with in vivo imaging of transparent embryos and larvae. The innate immune system has an essential role in the initial inflammatory response to viral infection. Focused studies of the innate immune response to viral infection are possible using the zebrafish model as there is a 4-6 week timeframe during development where they have a functional innate immune system dominated by neutrophils and macrophages. During this timeframe, zebrafish lack a functional adaptive immune system, so it is possible to study the innate immune response in isolation. Sequencing of the zebrafish genome has revealed significant genetic conservation with the human genome, and multiple studies have revealed both functional conservation of genes, including those critical to host cell infection and host cell inflammatory response. In addition to studying several fish viruses, zebrafish infection models have been developed for several human viruses, including influenza A, noroviruses, chikungunya, Zika, dengue, herpes simplex virus type 1, Sindbis, and hepatitis C virus. The development of these diverse viral infection models, coupled with the inherent strengths of the zebrafish model, particularly as it relates to our understanding of macrophage and neutrophil biology, offers opportunities for far more intensive studies aimed at understanding conserved host responses to viral infection. In this context, we review aspects relating to the evolution of innate immunity, including the evolution of viral pattern recognition receptors, interferons and interferon receptors, and non-coding RNAs.

Specific resistance to Pseudomonas aeruginosa infection in zebrafish is mediated by the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis (CF) is a genetic disease caused by recessive mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is associated with prevalent and chronic Pseudomonas aeruginosa lung infections. Despite numerous studies that have sought to elucidate the role of CFTR in the innate immune response, the links between CFTR, innate immunity, and P. aeruginosa infection remain unclear. The present work highlights the zebrafish as a powerful model organism for human infectious disease, particularly infection by P. aeruginosa. Zebrafish embryos with reduced expression of the cftr gene (Cftr morphants) exhibited reduced respiratory burst response and directed neutrophil migration, supporting a connection between cftr and the innate immune response. Cftr morphants were infected with P. aeruginosa or other bacterial species that are commonly associated with infections in CF patients, including Burkholderia cenocepacia, Haemophilus influenzae, and Staphylococcus aureus. Intriguingly, the bacterial burden of P. aeruginosa was found to be significantly higher in zebrafish Cftr morphants than in controls, but this phenomenon was not observed with the other bacterial species. Bacterial burden in Cftr morphants infected with a P. aeruginosa ΔLasR mutant, a quorum sensing-deficient strain, was comparable to that in control fish, indicating that the regulation of virulence factors through LasR is required for enhancement of infection in the absence of Cftr. The zebrafish system provides a multitude of advantages for studying the pathogenesis of P. aeruginosa and for understanding the role that innate immune cells, such as neutrophils, play in the host response to acute bacterial infections commonly associated with cystic fibrosis.

Broad-host-range plasmids for red fluorescent protein labeling of gram-negative bacteria for use in the zebrafish model system.

To observe real-time interactions between green fluorescent protein-labeled immune cells and invading bacteria in the zebrafish (Danio rerio), a series of plasmids was constructed for the red fluorescent protein (RFP) labeling of a variety of fish and human pathogens. The aim of this study was to create a collection of plasmids that would express RFP pigments both constitutively and under tac promoter regulation and that would be nontoxic and broadly transmissible to a variety of Gram-negative bacteria. DNA fragments encoding the RFP dimeric (d), monomeric (m), and tandem dimeric (td) derivatives d-Tomato, td-Tomato, m-Orange, and m-Cherry were cloned into the IncQ-based vector pMMB66EH in Escherichia coli. Plasmids were mobilized into recipient strains by conjugal mating. Pigment production was inducible in Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, and Vibrio (Listonella) anguillarum strains by isopropyl-beta-d-thiogalactopyranoside (IPTG) treatment. A spontaneous mutant exconjugant of P. aeruginosa PA14 was isolated that expressed td-Tomato constitutively. Complementation analysis revealed that the constitutive phenotype likely was due to a mutation in lacI(q) carried on pMMB66EH. DNA sequence analysis confirmed the presence of five transitions, four transversions, and a 2-bp addition within a 14-bp region of lacI. Vector DNA was purified from this constitutive mutant, and structural DNA sequences for RFP pigments were cloned into the constitutive vector. Exconjugants of P. aeruginosa, E. tarda, and V. anguillarum expressed all pigments in an IPTG-independent fashion. Results from zebrafish infectivity studies indicate that RFP-labeled pathogens will be useful for the study of real-time interactions between host cells of the innate immune system and the infecting pathogen.

Effects of low concentrations of arsenic on the innate immune system of the zebrafish (Danio rerio).

Arsenic has been associated with a multitude of human health problems; however, its impact on host resistance to infection has not been extensively researched. In vertebrates, the innate immune response is vital for potentiating the adaptive immune response. Therefore, dampening of the innate immune response results in an immunocompromised host. In this present study, effects of low concentrations of arsenic on zebrafish resistance to infection are evaluated. Exposure to 2 and 10 ppb arsenic, both considered safe levels in drinking water, resulted in a greater than 50-fold increase in viral load and at least a 17-fold increase in bacterial load in embryos. To determine the cause of this amplified pathogen load, important components of the innate immune system were analyzed. Presence of arsenic dampened the overall innate immune health of the fish as evidenced by reductions in respiratory burst activity. Viral infection, after arsenic exposure, showed decreases of up to 13- and 1.5-fold changes in interferon and Mx mRNA expression, respectively. Bacterial infection, post arsenic exposure, demonstrated at least 2.5- and 4-fold declines in interleukin-1beta and tumor necrosis factor-alpha mRNA levels, respectively. Maximum expression of these essential cytokines was also delayed upon arsenic exposure. Our data indicate that arsenic exposure, at concentrations deemed safe in drinking water, suppresses the overall innate immune function in zebrafish and present the zebrafish as a unique model for studying immunotoxicity of environmental toxicants. To our knowledge, this is the first report describing the effects of such low levels of arsenic on host resistance to infection.

Evolutionary divergence of the vertebrate TNFAIP8 gene family: Applying the spotted gar orthology bridge to understand ohnolog loss in teleosts.

Comparative functional genomic studies require the proper identification of gene orthologs to properly exploit animal biomedical research models. To identify gene orthologs, comprehensive, conserved gene synteny analyses are necessary to unwind gene histories that are convoluted by two rounds of early vertebrate genome duplication, and in the case of the teleosts, a third round, the teleost genome duplication (TGD). Recently, the genome of the spotted gar, a holostean outgroup to the teleosts that did not undergo this third genome duplication, was sequenced and applied as an orthology bridge to facilitate the identification of teleost orthologs to human genes and to enhance the power of teleosts as biomedical models. In this study, we apply the spotted gar orthology bridge to help describe the gene history of the vertebrate TNFAIP8 family. Members of the TNFAIP8 gene family have been linked to regulation of immune function and homeostasis and the development of multiple cancer types. Through a conserved gene synteny analysis, we identified zebrafish orthologs to human TNFAIP8L1 and TNFAIP8L3 genes and two co-orthologs to human TNFAIP8L2, but failed to identify an ortholog to human TNFAIP8. Through the application of the orthology bridge, we determined that teleost orthologs to human TNFAIP8 genes were likely lost in a genome inversion event after their divergence from their common ancestor with spotted gar. These findings demonstrate the value of this enhanced approach to gene history analysis and support the development of teleost models to study complex questions related to an array of biomedical issues, including immunity and cancer.

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses.

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Functional characterization of full-length TLR3, IRAK-4, and TRAF6 in zebrafish (Danio rerio).

Recently, the zebrafish, Danio rerio, has been recognized as a useful model for infectious disease and immunity. The Toll-like receptor (TLR) family is an evolutionarily conserved component of the innate immune system that responds to specific pathogen-associated molecular patterns (PAMPs) during an infection. This study reports the identification and characterization of a full-length orthologue of mammalian TLR3, and the key TLR pathway signaling molecules IRAK-4 and TRAF6 in the zebrafish. Sequence analysis of zebrafish TLR3 (zfTLR3), IRAK-4 (zfIRAK-4), and TRAF6 (zfTRAF6) revealed conserved domains shared with insect and mammalian genes. Quantitative real-time PCR showed that all three genes are expressed in a variety of adult tissues and during embryonic development. In in situ hybridization, we showed that zfTLR3, zfIRAK-4, and zfTRAF6 are present in distinct regions of the developing brain at 22hpf and that zfTRAF6 was observed in the developing medial neural tube. Overexpression of zfIRAK-4, zfTRAF6, or a mutant zfTLR3 construct was able to stimulate NF-kappaB activation in ZFL cells as measured by a cotransfected NF-kappaB-luciferase reporter plasmid. Messenger RNA expression profiles of each gene in zebrafish embryos and adults were examined by quantitative real-time PCR following infection with snakehead rhabdovirus (SHRV) or Edwardsiella tarda. Following exposure to SHRV, only zfTLR3 and zfTRAF6 mRNA transcripts were upregulated. Interestingly, exposure of fish to E. tarda resulted in an unexpected increase in mRNA expression of zfTLR3, as well as the anticipated upregulation of zfIRAK-4 and zfTRAF6 mRNA transcripts. These results demonstrate that zebrafish possess conserved TLR-signaling pathways, further emphasizing the utility of the zebrafish as a model for vertebrate immunology.

Pathogenesis and inflammatory response to Edwardsiella tarda infection in the zebrafish.

The zebrafish (Danio rerio) is a widely used model for developmental biology, neurobiology, toxicology, and genetic disease. Recently, the zebrafish has been recognized as a valuable model for infectious disease and immunity. In this study the pathogenesis and inflammatory cytokine response of zebrafish to experimental Edwardsiella tarda infection was characterized. In challenge experiments, zebrafish embryos were susceptible to infection by immersion. Adult fish were susceptible to challenge by intraperitoneal (ip) injection but not static immersion unless the epithelial layer was perturbed by scraping prior to exposure. To determine if E. tarda infection induces a typical acute inflammatory response, mRNA expression levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNFalpha) were assessed by quantitative real-time PCR. The expression levels of IL-1beta and TNFalpha were significantly upregulated in infected zebrafish embryos and adults. The methods developed in this study will be particularly valuable for targeted gene disruption studies of host immune components and in zebrafish genetic screens.

Effects of arsenic on zebrafish innate immune system.

The innate immune response, the first line of defense against invading pathogens, can be perturbed by environmental toxicants such as arsenic. This study reports the effects of arsenic on innate immunity of zebrafish. Respiratory burst activity, messenger RNA expression of tumor necrosis factor alpha (TNF-alpha), a primer of the respiratory burst response, and mRNA expression of the antiviral cytokines interferon (IFN) and MX, : before and after viral infection, were examined in arsenic-exposed zebrafish larvae. Respiratory burst activity and TNF-alpha expression were decreased upon arsenic exposure, indicating inhibition of TNF-alpha priming of the respiratory burst response. Arsenic enhanced IFN expression slightly over time, but reduced MX : expression. In zebrafish infected with snakehead rhabdovirus, arsenic decreased induction and altered the kinetics of IFN and MX : upon infection. Differences in IFN and MX : expression in arsenic-exposed larvae point toward an interruption of the Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathway.

Nanoscale imaging of caveolin-1 membrane domains in vivo.

Light microscopy enables noninvasive imaging of fluorescent species in biological specimens, but resolution is generally limited by diffraction to ~200-250 nm. Many biological processes occur on smaller length scales, highlighting the importance of techniques that can image below the diffraction limit and provide valuable single-molecule information. In recent years, imaging techniques have been developed which can achieve resolution below the diffraction limit. Utilizing one such technique, fluorescence photoactivation localization microscopy (FPALM), we demonstrated its ability to construct super-resolution images from single molecules in a living zebrafish embryo, expanding the realm of previous super-resolution imaging to a living vertebrate organism. We imaged caveolin-1 in vivo, in living zebrafish embryos. Our results demonstrate the successful image acquisition of super-resolution images in a living vertebrate organism, opening several opportunities to answer more dynamic biological questions in vivo at the previously inaccessible nanoscale.

Development of a respiratory burst assay using zebrafish kidneys and embryos.

The innate immune response constitutes the first line of defense against invading pathogens and consists of a variety of immune defense mechanisms including the respiratory burst of phagocytes. Respiratory burst can be used as a reliable measure of the immune response of a host, and numerous assays have been developed to measure this response in a variety of mammal and fish species. Phagocytes, like granulocytes and macrophages, that are derived from different tissues, or grown in cell culture, have been employed in a range of assay formats employing a variety of detection methods. The small size of the zebrafish has prevented the large-scale extraction of these cells for respiratory burst assays in the zebrafish. In this work, we describe a respiratory burst assay developed for the zebrafish using intact kidneys and embryos as sources of phagocytes. Phorbol myristate acetate (PMA)-inducible reactive oxygen species (ROS) were detected following the oxidation of a non-fluorescent dye 2',7'-dihydrodichlorofluorescein diacetate (H2DCFDA) to dichlorofluorescein (DCF), a fluorescent product. Embryos from 1 day post-fertilization until 5 days post-fertilization (dpf) were employed in this assay. Abrogation of H2DCFDA oxidation by the protein kinase C (PKC) inhibitor bisindolylmaleimide I (BisI) indicated a reduction in the respiratory burst. Fluorescence from the PMA-induced respiratory burst in kidneys and embryos was significantly elevated above DMSO-treated controls, while preincubation with BisI inhibited the increase in fluorescence. Colocalization of cell-associated chloromethyl-dihydrodichlorofluorescein diacetate (CM-H2DCFDA) with the phagocyte-selective dye neutral red is consistent with the observation that macrophages and granulocytes are the ROS-producing cells in the zebrafish.

Cloning and characterization of an Mx gene and its corresponding promoter from the zebrafish, Danio rerio.

Type I interferons (IFNs) represent a crucial component of the innate immune response to viruses. An important downstream effector of IFN is the Mx gene, which is activated solely through this pathway. Mx proteins are characterized by a tripartite GTP-binding domain, dynamin family signature, and leucine zipper motif. Mx genes are transcribed upon activation of an interferon-stimulated response element (ISRE) located in the Mx promoter region. In this article, we describe the cloning and analysis of an Mx gene and its corresponding promoter from the zebrafish (Danio rerio). The deduced amino acid sequence of zebrafish Mx contains the conserved GTP-binding domain, dynamin family signature, and leucine zipper motif common to Mx proteins, and shows a 50% identity to human MxA and 69% identity both to rainbow trout and to Atlantic salmon. Zebrafish liver cells produced high levels of Mx mRNA in response to induction by the known IFN-inducer polyinosinic-polycytidylic acid (Poly[I:C]). The zebrafish Mx promoter contains two ISREs homologous to those found in the promoter regions of many IFN-inducible genes, and was able to drive transcription of a luciferase reporter gene when induced by either purified zebrafish IFN or Poly[I:C].

Studying the immune response to human viral infections using zebrafish.

Humans and viruses have a long co-evolutionary history. Viral illnesses have and will continue to shape human history: from smallpox, to influenza, to HIV, and beyond. Animal models of human viral illnesses are needed in order to generate safe and effective antiviral medicines, adjuvant therapies, and vaccines. These animal models must support the replication of human viruses, recapitulate aspects of human viral illnesses, and respond with conserved immune signaling cascades. The zebrafish is perhaps the simplest, most commonly used laboratory model organism in which innate and/or adaptive immunity can be studied. Herein, we will discuss the current zebrafish models of human viral illnesses and the insights they have provided. We will highlight advantages of early life stage zebrafish and the importance of innate immunity in human viral illnesses. We will also discuss viral characteristics to consider before infecting zebrafish with human viruses as well as predict other human viruses that may be able to infect zebrafish.

Influenza A virus infection in zebrafish recapitulates mammalian infection and sensitivity to anti-influenza drug treatment.

Seasonal influenza virus infections cause annual epidemics and sporadic pandemics. These present a global health concern, resulting in substantial morbidity, mortality and economic burdens. Prevention and treatment of influenza illness is difficult due to the high mutation rate of the virus, the emergence of new virus strains and increasing antiviral resistance. Animal models of influenza infection are crucial to our gaining a better understanding of the pathogenesis of and host response to influenza infection, and for screening antiviral compounds. However, the current animal models used for influenza research are not amenable to visualization of host-pathogen interactions or high-throughput drug screening. The zebrafish is widely recognized as a valuable model system for infectious disease research and therapeutic drug testing. Here, we describe a zebrafish model for human influenza A virus (IAV) infection and show that zebrafish embryos are susceptible to challenge with both influenza A strains APR8 and X-31 (Aichi). Influenza-infected zebrafish show an increase in viral burden and mortality over time. The expression of innate antiviral genes, the gross pathology and the histopathology in infected zebrafish recapitulate clinical symptoms of influenza infections in humans. This is the first time that zebrafish embryos have been infected with a fluorescent IAV in order to visualize infection in a live vertebrate host, revealing a pattern of vascular endothelial infection. Treatment of infected zebrafish with a known anti-influenza compound, Zanamivir, reduced mortality and the expression of a fluorescent viral gene product, demonstrating the validity of this model to screen for potential antiviral drugs. The zebrafish model system has provided invaluable insights into host-pathogen interactions for a range of infectious diseases. Here, we demonstrate a novel use of this species for IAV research. This model has great potential to advance our understanding of influenza infection and the associated host innate immune response.

Quantification of the respiratory burst response as an indicator of innate immune health in zebrafish.

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.