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      Whole-Body Analysis of a Viral Infection: Vascular Endothelium is a Primary Target of Infectious Hematopoietic Necrosis Virus in Zebrafish Larvae

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          Abstract

          The progression of viral infections is notoriously difficult to follow in whole organisms. The small, transparent zebrafish larva constitutes a valuable system to study how pathogens spread. We describe here the course of infection of zebrafish early larvae with a heat-adapted variant of the Infectious Hematopoietic Necrosis Virus (IHNV), a rhabdovirus that represents an important threat to the salmonid culture industry. When incubated at 24°C, a permissive temperature for virus replication, larvae infected by intravenous injection died within three to four days. Macroscopic signs of infection followed a highly predictable course, with a slowdown then arrest of blood flow despite continuing heartbeat, followed by a loss of reactivity to touch and ultimately by death. Using whole-mount in situ hybridization, patterns of infection were imaged in whole larvae. The first infected cells were detectable as early as 6 hours post infection, and a steady increase in infected cell number and staining intensity occurred with time. Venous endothelium appeared as a primary target of infection, as could be confirmed in fli1:GFP transgenic larvae by live imaging and immunohistochemistry. Disruption of the first vessels took place before arrest of blood circulation, and hemorrhages could be observed in various places. Our data suggest that infection spread from the damaged vessels to underlying tissue. By shifting infected fish to a temperature of 28°C that is non-permissive for viral propagation, it was possible to establish when virus-generated damage became irreversible. This stage was reached many hours before any detectable induction of the host response. Zebrafish larvae infected with IHNV constitute a vertebrate model of an hemorrhagic viral disease. This tractable system will allow the in vivo dissection of host-virus interactions at the whole organism scale, a feature unrivalled by other vertebrate models.

          Author Summary

          The zebrafish larva is uniquely amenable to imaging among vertebrate models because of its small size, transparency, and ease of anesthesia, making it a useful model to understand host-pathogen interactions. We have performed the first detailed analysis of a viral infection in zebrafish. Infection of zebrafish larvae with a salmonid rhabdovirus adapted to growth at the appropriate temperatures resulted in a predictable succession of pathological signs before death. Detection of infected cells in whole larvae revealed that blood vessels were a major target of the virus, providing an explanation to hemorrhages and subsequent loss of blood flow observed in infected larvae. Destruction of vascular cells caused by the viral infection was readily observed in transgenic larvae with fluorescent endothelium. We could identify the critical moments of the infection with simple temperature shift experiments. This work provides a basis to dissect the role of host factors in controlling the propagation of viral infections.

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          Most cited references31

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          Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants.

          The zebrafish is firmly established as a genetic model for the study of vertebrate blood development. Here we have characterized the blood-forming system of adult zebrafish. Each major blood lineage can be isolated by flow cytometry, and with these lineal profiles, defects in zebrafish blood mutants can be quantified. We developed hematopoietic cell transplantation to study cell autonomy of mutant gene function and to establish a hematopoietic stem cell assay. Hematopoietic cell transplantation can rescue multilineage hematopoiesis in embryonic lethal gata1-/- mutants for over 6 months. Direct visualization of fluorescent donor cells in embryonic recipients allows engraftment and homing events to be imaged in real time. These results provide a cellular context in which to study the genetics of hematopoiesis.
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            The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development.

            We have used confocal microangiography to examine and describe the vascular anatomy of the developing zebrafish, Danio rerio. This method and the profound optical clarity of zebrafish embryos make it possible to view the entire developing vasculature with unprecedented resolution. A staged series of three-dimensional images of the vascular system were collected beginning shortly after the onset of circulation at 1 day postfertilization through early- to midlarval stages at approximately 7 days postfertilization. Blood vessels in every region of the animal were imaged at each stage, and detailed "wiring patterns" were derived describing the interconnections between every major vessel. We present an overview of these data here in this paper and in an accompanying Web site "The interactive atlas of zebrafish vascular anatomy" online at (http://eclipse.nichd.nih.gov/nichd/lmg/redirect.html). We find a highly dynamic but also highly stereotypic pattern of vascular connections, with different sets of primitive embryonic vessels severing connections and rewiring in new configurations according to a reproducible plan. We also find that despite variation in the details of the vascular anatomy, the basic vascular plan of the developing zebrafish shows strong similarity to that of other vertebrates. This atlas will provide an invaluable foundation for future genetic and experimental studies of vascular development in the zebrafish.
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              Ontogeny and behaviour of early macrophages in the zebrafish embryo.

              In the zebrafish embryo, the only known site of hemopoieisis is an intra-embryonic blood island at the junction between trunk and tail that gives rise to erythroid cells. Using video-enhanced differential interference contrast microscopy, as well as in-situ hybridization for the expression of two new hemopoietic marker genes, draculin and leucocyte-specific plastin, we show that macrophages appear in the embryo at least as early as erythroid cells, but originate from ventro-lateral mesoderm situated at the other end of the embryo, just anterior to the cardiac field. These macrophage precursors migrate to the yolksac, and differentiate. From the yolksac, many invade the mesenchyme of the head, while others join the blood circulation. Apart from phagocytosing apoptotic corpses, these macrophages were observed to engulf and destroy large amounts of bacteria injected intravenously; the macrophages also sensed the presence of bacteria injected into body cavities that are isolated from the blood, migrated into these cavities and eradicated the microorganisms. Moreover, we observed that although only a fraction of the macrophage population goes to the site of infection, the entire population acquires an activated behaviour, similar to that of activated macrophages in mammals. Our results support the notion that in vertebrate embryos, macrophages endowed with proliferative capacity arise early from the hemopoietic lineage through a non-classical, rapid differentiation pathway, which bypasses the monocytic series that is well-documented in adult hemopoietic organs.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                February 2011
                February 2011
                3 February 2011
                : 7
                : 2
                : e1001269
                Affiliations
                [1 ]Macrophages et Développement de l'Immunité, Institut Pasteur, Paris, France
                [2 ]CNRS URA2578, Paris, France
                [3 ]Virologie et Immunologie Moléculaire, INRA, Jouy-en-Josas, France
                University of Washington, United States of America
                Author notes

                ¤: Current address: Department of Cardiac Surgery, University of Rostock, Rostock, Germany.

                Conceived and designed the experiments: ECG PB JPL. Performed the experiments: ML NP CT VB JPL. Analyzed the data: ML NP ECG PH PB JPL. Contributed reagents/materials/analysis tools: MB. Wrote the paper: PB JPL.

                Article
                10-PLPA-RA-3702R2
                10.1371/journal.ppat.1001269
                3033377
                21304884
                ea9d6a97-8022-40fe-977a-7fb1fc8417c3
                Ludwig et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 2 July 2010
                : 3 January 2011
                Page count
                Pages: 11
                Categories
                Research Article
                Immunology/Innate Immunity
                Infectious Diseases/Viral Infections
                Virology/Animal Models of Infection
                Virology/Host Antiviral Responses
                Virology/Host Invasion and Cell Entry

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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