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.

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.

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.

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.

Monitoring polymer properties using shear horizontal surface acoustic waves.

Real-time, nondestructive methods for monitoring polymer film properties are increasingly important in the development and fabrication of modern polymer-containing products. Online testing of industrial polymer films during preparation and conditioning is required to minimize material and energy consumption, improve the product quality, increase the production rate, and reduce the number of product rejects. It is well-known that shear horizontal surface acoustic wave (SH-SAW) propagation is sensitive to mass changes as well as to the mechanical properties of attached materials. In this work, the SH-SAW was used to monitor polymer property changes primarily dictated by variations in the viscoelasticity. The viscoelastic properties of a negative photoresist film were monitored throughout the ultraviolet (UV) light-induced polymer cross-linking process using SH-SAW delay line devices. Changes in the polymer film mass and viscoelasticity caused by UV exposure produced variations in the phase velocity and attenuation of the SH-SAW propagating in the structure. Based on measured polymer-coated delay line scattering transmission responses (S(21)) and the measured polymer layer thickness and density, the viscoelastic constants c(44) and eta(44) were extracted. The polymer thickness was found to decrease 0.6% during UV curing, while variations in the polymer density were determined to be insignificant. Changes of 6% in c(44) and 22% in eta(44) during the cross-linking process were observed, showing the sensitivity of the SH-SAW phase velocity and attenuation to changes in the polymer film viscoelasticity. These results indicate the potential for SH-SAW devices as online monitoring sensors for polymer film processing.

Evidence for evolving Toll-IL-1 receptor-containing adaptor molecule function in vertebrates.

In mammals, Toll-IL-1R-containing adaptor molecule 1 (TICAM1)-dependent TLR pathways induce NF-kappaB and IFN-beta responses. TICAM1 activates NF-kappaB through two different pathways involving its interactions with TNFR-associated factor 6 and receptor-interacting protein 1. It also activates IFN regulatory factor 3/7 through its interaction with TANK-binding kinase-1, leading to the robust up-regulation of IFN-beta. In this study, we describe the role of zebrafish (Danio rerio) TICAM1 in activating NF-kappaB and zebrafish type I IFN. Zebrafish IFN is unique in that it cannot be categorized as being alpha- or beta-like. Through comprehensive sequence, phylogenetic, and syntenic analyses, we fully describe the identification of a zebrafish TICAM1 ortholog. Zebrafish TICAM1 exhibits sequence divergence from its mammalian orthologs and our data demonstrate that these sequence differences have functional consequences. Zebrafish TICAM1 activates zebrafish IFN; however, it does so in an apparently IFN regulatory factor 3/7-independent manner. Furthermore, zebrafish TICAM1 does not interact with zebrafish TNFR-associated factor 6, thus NF-kappaB activation is dependent upon its interaction with receptor-interacting protein 1. Comparative genome analysis suggests that TICAM1 and TICAM2 evolved from a common vertebrate TICAM ancestor following a gene duplication event and that TICAM2 was lost in teleosts following the divergence of the rayfin and lobefin fishes 450 million years ago. These studies provide evidence, for the first time, of the evolving function of a vertebrate TLR pathway.

Nucleic acid sensing by regenerable surface-associated isothermal rolling circle amplification.

A novel method for regenerating biosensors has been developed in which the highly specific detection of nucleic acid sequences is carried out using molecular padlock probe (MPP) technology and surface-associated rolling circle amplification (RCA). This technique has a low occurrence of false positive results when compared to polymerase chain reaction, and is an isothermal reaction, which is advantageous in systems requiring low power consumption such as remote field sensing applications. Gold-sputtered 96-well polystyrene microplates and a fluorescent label were used to explore the detection limits of the surface-associated RCA technique, specificity for different MPP, conditions for regeneration of the biomolecular sensing surface, and reproducibility of measurements on regenerated surfaces. The technique was used to create highly selective biomolecular surfaces capable of discriminating between DNA oligonucleotides with sequences identical to RNA from infectious salmon anemia (ISA) and infectious hematopoietic necrosis (IHN) virus. As little as 0.6 fmol of circularized MPP was detectable with this fluorimetric assay. The sensing layers could be reused for at least four cycles of amplification using thermal denaturation, with less than 33% decrease in RCA response over time. Because the nucleic acid product of the test is attached to a surface during amplification, the technique is directly applicable to a variety of existing sensing platforms, including acoustic wave and optical devices.

Detection and identification of IHN and ISA viruses by isothermal DNA amplification in microcapillary tubes.

Unique base sequences derived from RNA of both infectious hematopoietic necrosis virus (IHNV) and infectious salmon anemia virus (ISAV) were detected and identified using a combination of surface-associated molecular padlock DNA probes (MPPs) and rolling circle amplification (RCA) in microcapillary tubes. DNA oligonucleotides with base sequences identical to RNA obtained from IHNV or ISAV were recognized by MPPs. Circularized MPPs were then captured on the inner surfaces of glass microcapillary tubes by immobilized DNA oligonucleotide primers. Extension of the immobilized primers by isothermal RCA produced DNA concatamers, which were labeled with fluorescent SYBR Green II nucleic acid stain, and measured by microfluorimetry. Molecular padlock probes, combined with this method of surface-associated isothermal RCA, exhibited high selectivity without the need for thermal cycling. This method is applicable to the design of low-power field sensors capable of multiplex detection of viral, bacterial, and protozoan pathogens within localized regions of microcapillary tubes.

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.