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Cellular Visualization of Macrophage Pyroptosis
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and IL1 Release in a Viral Hemorrhagic
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Infection in Zebrafish Larvae
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Mónica Varela1, Alejandro Romero1, Sonia Dios1, Michiel van der Vaart2, Antonio
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Figueras1, Annemarie H. Meijer2, Beatriz Novoa1#
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1 Marine Research Institute, CSIC, Vigo, Spain, 2 Institute of Biology, Leiden University, Leiden, The
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Netherlands
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Corresponding Author: beatriznovoa@iim.csic.es
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Running Tittle: Macrophage Pyroptosis in Zebrafish Larvae
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Keywords: Hemorrhagic Virus, Zebrafish, Macrophages, Pyroptosis, IL1 Release, Antiviral Response,
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RNA Virus, Inflammatory Response, SVCV
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Abstract word count: 284
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Text word count: 5097
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ABSTRACT
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Hemorrhagic viral diseases are distributed worldwide with important pathogens,
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such as Dengue virus or Hantaviruses. The lack of adequate in vivo infection models has
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limited the research on viral pathogenesis and the current understanding of the
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underlying infection mechanisms. Although hemorrhages have been associated with the
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infection of endothelial cells, other cellular types could be the main targets for
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hemorrhagic viruses. Our objective was to take advantage of the use of zebrafish larvae
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in the study of viral hemorrhagic diseases, focusing on the interaction between viruses
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and host cells. Cellular processes, such as transendothelial migration of leukocytes,
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viral-induced pyroptosis of macrophages and Il1 release, could be observed in
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individual cells, providing a deeper knowledge of the immune mechanisms implicated
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in the disease. Furthermore, the application of these techniques to other pathogens will
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improve the current knowledge of host-pathogen interactions and increase the potential
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for the discovery of new therapeutic targets.
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IMPORTANCE
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Pathogenic mechanisms of hemorrhagic viruses are diverse and most of the
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research regarding interactions between viruses and host cells has been performed in
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cell lines that might not be major targets during natural infections. Thus, viral
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pathogenesis research has been limited because of the lack of adequate in vivo infection
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models. The understanding of the relative pathogenic roles of the viral agent and the
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host response to the infection is crucial. This will be facilitated by the establishment of
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in vivo infection models using organisms as zebrafish, which allows the study of the
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diseases in the context of complete individual. The use of this animal model with other
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pathogens could improve the current knowledge on host-pathogen interactions and
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increase the potential for the discovery of new therapeutic targets against diverse viral
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diseases.
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INTRODUCTION
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Endothelial cells and leukocytes are at the front line of defense against
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pathogens. Most infectious pathogens have some relationship with the endothelium,
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although not all pathogens are true endothelial invading organisms (1). Infections can
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produce large changes in endothelial cells function, particularly in relation to
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inflammation. Inflammatory stimuli can activate endothelial cells, which secrete
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cytokines and chemokines. Moreover, the importance of endothelial cells in the
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regulation of leukocyte transmigration to the site of inflammation has given these cells a
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major role in immunity (2).
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Host-pathogen interactions are essential for the modulation of immunity (3). The
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presence of a pathogen alters the immune balance prevailing in a host under normal
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conditions causing the appearance of diseases, which lead to the death of the host if it is
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not able to control the infection. Clear examples of these effects are evident in viral
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hemorrhagic fevers, in which an understanding of the relative pathogenic roles of the
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viral agent and the host response to the infection is crucial (4). Hemorrhagic
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manifestations are characteristic of these fevers, which have historically been associated
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with endothelium infections. Currently, many pathogens causing hemorrhagic diseases
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do not infect endothelial cells as the primary target (1). Indeed, macrophages and
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dendritic cells are targets for some hemorrhagic viruses, such as Ebola Virus (5) or
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Dengue Virus (6, 7).
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Most of the research regarding interactions between viruses and host cells has
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been performed in cell lines that might not be major targets during natural infections
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(8), making it difficult to characterize the behavior of pathogens in individuals and
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reducing the likelihood of discovering an effective therapeutic target. The lack of a good
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animal model in which the infection can be followed from the beginning and in the
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context of the whole organism has contributed to these difficulties. Zebrafish (Danio
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rerio) provide advantages, as these organisms exhibit rapid development, transparency
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and a high homology between its genome and the human one (9). Furthermore, the
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existence of mutant and transgenic fish lines and the potential use of reverse genetics
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techniques, such as the injection of antisense morpholino oligonucleotides or synthetic
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mRNA, make zebrafish a useful tool for the study of host-pathogen interactions.
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No naturally occurring viral infections have been characterized for zebrafish
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(10). Moreover, the experimental susceptibility of zebrafish to viral infections has been
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demonstrated with other fish viruses (11-13) and also with mammalian viruses (14-16).
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We used the rhabdovirus Spring Viraemia of Carp Virus (SVCV) as a viral
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model to show the potential of the zebrafish in host-pathogen interaction studies. SVCV
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predominantly affects cyprinid fish and causes important economical losses and
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mortalities worldwide (17, 18). Affected fish exhibit destruction of tissues, such as
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kidney or spleen, leading to hemorrhage and death. However, little is known about the
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infection mechanism of this enveloped RNA virus.
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Thus, the main objective of the present study was to improve the knowledge of
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the pathology generated by SVCV, as an example of hemorrhagic virus, focusing on
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interactions between the virus and host cells at early stages of the infection in zebrafish
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larvae. Taking advantage of zebrafish transgenic lines, imaging techniques and, using
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morpholino-induced cell depletion, viral-induced pyroptosis and IL1β release were
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observed in macrophages at the single cell level.
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MATERIALS AND METHODS
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Ethics Statement. The protocols for fish care and the challenge experiments were
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reviewed and approved by the CSIC National Committee on Bioethics under approval
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number 07_09032012. Experiments were conducted in fish larvae before independently
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feeding and therefore, before an ethical approval is required (EU directive 2010_63).
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Fish. Homozygous embryos and larvae from wild-type zebrafish, transgenic line
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Tg(Fli1a:eGFP)y1 that labels endothelial cells, transgenic line Tg(Mpx:GFP)i114 that
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labels neutrophils, transgenic line Tg(Mpeg1:eGFP)gl22 that labels macrophages and
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transgenic line Tg(Cd41:GFP)la2 that labels thrombocytes were obtained from our
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experimental facility, where the zebrafish were cultured using established protocols (19,
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20). The eggs were obtained according to protocols described in The Zebrafish Book
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(19) and maintained at 28.5˚C in egg water (5 mM NaCl, 0.17 mM KCl, 0.33 mM
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CaCl2, 0.33 mM MgSO4, and 0.00005% methylene blue), and 0.2 mM N-
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phenylthiourea (PTU; Sigma) was used to prevent pigment formation from 1 days post-
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fertilization (dpf).
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Virus and Infection. The SVCV isolate 56/70 was propagated on epithelioma
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papulosum cyprini (EPC) carp cells (ATCC CRL-2872) and titrated in 96-well plates.
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The plates were incubated at 15°C for 7 days and examined for cytophathic effects each
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day. After the observation of the cells under the microscope, the virus dilution causing
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infection of 50% of the inoculated cell line (TCID50) was determined using the Reed-
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Müench method (21). The fish larvae were infected through microinjection into the duct
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of Cuvier as described in Benard et al. (22) at 2 or 3 dpf. SVCV was diluted to the
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appropriate concentration in PBS with 0.1% phenol red (as a visible marker to the
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injection of the solution into the embryo) just before the microinjection of 2 nl of viral
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suspension per larvae. The infections were conducted at 23˚C. SVCV was heat-killed at
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65ºC during 20 minutes.
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Imaging. Images of the signs and videos of the blood flow were obtained using an
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AZ100 microscope (Nikon) coupled to a DS-Fi1 digital camera (Nikon). Confocal
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images of live or fixed larvae were captured using a TSC SPE confocal microscope
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(Leica). The images were processed using the LAS-AF (Leica) and ImageJ software.
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The 3D reconstructions and the volume clipping were performed using Image Surfer
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(http://imagesurfer.cs.unc.edu/) and LAS-AF (Leica), respectively.
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Immunohistochemistry. Whole-mount immunohistochemistry was performed as
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previously described (23). The following primary antibodies and dilutions were used: L-
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plastin (rabbit anti-zebrafish, 1:500, kindly provided by Dr. Paul Martin); Caspase a
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(rabbit anti-zebrafish, 1:500; Anaspec); SVCV (mouse anti-SVCV, 1:500; BioX
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Diagnostics) and Il1β (rabbit anti-zebrafish, 1:500, kindly provided by Dr. John
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Hansen). The secondary antibodies used were Alexa 488 anti-rabbit, Alexa 546 anti-
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rabbit or Alexa 635 anti-mouse (Life Technologies), all diluted 1:1000.
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Real-Time PCR. Total RNA was isolated from snap-frozen larvae using the Maxwell®
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16 LEV simply RNA Tissue kit (Promega) with the automated Maxwell® 16
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Instrument according to the manufacturer’s instructions. cDNA synthesis using random
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primers and qPCR were performed as previously described (24). 18S was used as a
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housekeeping to normalize the expression values. Gene expression ratio was calculated
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by dividing the normalized expression values of infected larvae by the normalized
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expression values of the controls. Table S1 in supplemental material shows the
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sequences of the primer pairs used. Two independent experiments, of 3 biological
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replicates each, were performed.
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Microangiography. Tetramethylrhodamine dextran (2x106 MW, Life Technologies)
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was injected into the caudal vein of anesthetized zebrafish Tg(Fli1a-GFP) embryos at
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24 hours post-infection (hpi). The images were acquired within 30 min after injection.
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TUNEL assay. The TUNEL assay was performed using the In Situ Cell Death
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Detection Kit, TMR red or POD (Roche) as previously described (25).
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In vivo acridine orange staining. For acridine orange staining, larvae were incubated
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for 2 hours in 10 µg/ml acridine orange solution followed by washing for 30 min.
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Macrophages depletion with PU.1-MO. For the PU.1 knockdown, the morpholino
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was injected into the yolk at the one-cell stage at 1 mM to block macrophages
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development as previously described (26). Macrophage-depleted fish were infected at 2
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dpf to ensure morpholino effectiveness until the end of the infection.
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In vivo propidium iodide staining. For propidium iodide (PI) staining, larvae were
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incubated for 2 hours in 10 µg/ml propidium iodide solution followed by washing for 30
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min.
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Statistical analysis. Kaplan-Meier survival curves were analyzed with a Log-rank
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(Mantel Cox) test. The neutrophil count and qPCR data were analyzed using one-way
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analysis of variance and Tukey’s test, and differences were considered significant at
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p<0.05. The results are presented as the means ± standard error of mean (SEM).
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RESULTS
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Course of the SVCV infection in zebrafish larvae. Zebrafish larvae are susceptible to
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SVCV through bath infection, but typically the results show high interindividual
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variations (12, 27). Moreover, the long infection times required exclude the use of
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temporal knockdown techniques, such as morpholino microinjection. As with caudal
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vein injection (27), we found that SVCV injection into the duct of Cuvier was efficient,
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thereby providing a reproducible route of infection with fast kinetics. We injected 2 nl
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of a SVCV stock solution (3x106 TCID50/ml) per larva. Mortality began at 22-24 hpi,
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consistently reaching nearly 100% between 48 and 50 hpi (Fig. 1A). There were no
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differences in the development of the infection between the use of 2 or 3 dpf larvae
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(data not shown). To assess viral transcription, the expression of the SVCV
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nucleoprotein (N gene) was measured through quantitative RT-PCR (Fig. 1B). The
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samples were collected between 3 and 24 hpi (just prior to mortality). Efficient viral
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transcription was observed from 6 hpi, with an important increment between 12 and 18
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hpi. Moreover, we found that viral N gene transcription began quickly in the fish as
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significant differences were found between heat-killed SVCV (HK-SVCV) and SVCV
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infected fish (Fig. 1B).
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With regard to macroscopic signs, the first ones were visible after 18 hours (Fig.
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1C). A progressive decrease in the blood flow rate was observed, particularly in the tail
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(See movie S1-S2 in supplemental material). The circulation in the tail was completely
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stopped after 20-22 hpi, although it remained visible at the anterior part of the fish. The
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circulation in the head lasted longer but also eventually stopped (See movie S3 in
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supplemental material). At this point, blood was clearly visible inside the veins, with
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exception of the hemorrhage areas (Fig. 1C). Hemorrhages appeared in the anterior part
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of the larvae, mostly in areas with a huge amount of microvessels but, a great
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heterogeneity was observed in the occurrence of these hemorrhages varying in terms of
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location and time post-infection. The heartbeat was clearly less powerful over time until
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it completely stopped, moment in which the larvae were considered dead. The observed
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signs and times are consistent with those previously described for this virus using a
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similar infection model (27). Consistency in the infection results reaffirms the idea that
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SVCV and zebrafish larvae are an appropriate duo for an in-depth study of the innate
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immune response against that intracellular pathogen.
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SVCV does not affect the integrity of the endothelium. The loss of circulation in the
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trunk and hemorrhages in larvae head were the most characteristic symptoms of
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infection. Therefore, we characterized the effect of the virus in the vascular system of
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the fish, as previously determined for another rhabdovirus, IHNV (13). Using the
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transgenic line Tg(Fli1a-GFP) (28), which facilitates the visualization of the vascular
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system, we clearly observed a loss of GFP fluorescence in infected fish at 24 hpi (Fig.
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1D, E). This effect was particularly visible in the main veins of the head (Fig. 1D) and
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in the tail (Figure 1E). In addition, we used this transgenic line in combination with a
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whole-mount fluorescent immunohistochemistry for L-plastin (leukocyte-plastin) (29).
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The anti-L-plastin antibody was a useful tool for the detection of macrophages and
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neutrophils in the larvae. In addition to the loss of Fli1a-GFP fluorescence in the trunk,
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a decrease in the number of L-plastin-positive cells in the area corresponding to the
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caudal hematopoietic tissue was also observed at 24 hpi (Fig. 1E). Moreover, a shape
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change in L-plastin-positive cells was clearly visible in infected fish. The morphology
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of the cells in infected fish changed from thin and elongated (with prolonged
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extensions) to rounded and lobulated (Fig. 1F). The morphological changes might be
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associated with alterations in cell motility or the infection of the leukocytes themselves
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(30, 31).
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To confirm the integrity of the endothelium in the tail at 24 hpi, we injected
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tetramethylrhodamine dextran into the caudal vein of the larvae. This fluorescent dye
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remained inside the vessels, even when Fli1a-GFP fluorescence was absent (Fig. 2A).
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Shadows in tetramethylrhodamine dextran fluorescence correspond with blood cells that
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remain inside vessels even when circulation was stopped (Fig. 2A, 2B). Moreover, the
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results of the TUNEL assay and the acridine orange staining revealed no DNA damage
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neither necrosis, respectively, in the endothelial cells of infected fish (Fig. 2C),
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suggesting that the cells were apparently healthy and the loss of fluorescence likely
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reflected a transcriptional regulation of the Fli1a gene and not cell death. This was
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verified by combining Fli1a-GFP fish with DIC microscopy (Fig. 2D). Further,
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TUNEL-positive cells in the caudal hematopoietic tissue of infected larvae were
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observed, and the localization of these cells was consistent with the position of L-
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plastin-positive hematopoietic precursor cells in control fish (Fig. 2E). This observation
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was not surprising as blood flow is essential for hematopoietic stem cell development
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(32). In addition, several studies have shown the relationship between blood circulation
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and Fli1, as Fli1-deficient mice exhibit impaired hematopoiesis (33). Indeed, previous
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studies in mice described that both morphants and knockouts of Fli1 showed normal
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vessel development, with impaired circulation and hemorrhaging (33, 34). This might
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suggest that the simple transcriptional regulation of fli1a could be sufficient to cause the
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symptoms observed in SVCV-infected larvae.
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To determine the mechanism underlying fli1a mRNA transcription and to
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associate it with the loss of blood flow in the larvae, we performed a time course during
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SVCV infection. Based on the signs of their morphants and on their implication in the
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maintenance of vessel integrity, we also selected the v-ets erythroblastosis virus E26
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oncogene homolog 1a (ets1a) and the vascular endothelial cadherin (ve-cad) genes for
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qPCR analysis (Fig. 2F). Fli1a and Ets1a are both transcription factors involved in the
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regulation of hematopoiesis and vasculogenesis (35). A statistically significant decrease
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in the level of fli1a gene transcripts was observed from 12 hpi. Similar results were
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observed for ets1a, as the time course profile for this transcription factor was identical
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to that for fli1a. The interindividual variations in ve-cad, a key regulator of endothelial
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intercellular junctions (36), made it difficult to observe a clear expression pattern,
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particularly at the late stages of the infection. This effect might be associated with the
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observed heterogeneity in the appearance of hemorrhages, in terms of time, area or
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larvae. Notably, the downregulation of ve-cad at 9 hpi suggests an increase in the
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permeability of the endothelium at that infection point. Interestingly, fli1a
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downregulation clearly occurred before the onset of hemorrhages and the loss of
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circulation, suggesting that hemorrhages were a consequence of this downregulation.
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Zebrafish antiviral response to SVCV. To further study the mechanisms involved in
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the defense against SVCV, we analyzed the expression of some important genes
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associated with viral detection. Pattern recognition receptors, such as Toll-like receptors
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(TLRs) and RIG-1-like receptors (RLRs), are membrane-bound and cytosolic sensors,
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respectively (37). We observed a significant downregulation of tlr7, tlr22, tlr8a and
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mda5 at 3 hpi (Fig. 3A). As occurred with Dengue virus (38) or Influenza (39), this
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downmodulation could correspond to an immune evasion strategy. rig-1 was not
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regulated during the first 24 hours of the infection. Regarding tlr3, we also saw a slight,
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but significant, regulation at 12 hpi.
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Pattern recognition receptors initiate antimicrobial defense mechanisms through several
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conserved signaling pathways (40). The activation of interferon-regulatory factors
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induces the production of several antiviral response-related genes. We observed that
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SVCV elicited a response in all the analyzed genes (Fig. 3B). ifnphi1 and ifnphi2
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showed different response profiles. ifnphi1 remained downregulated between 3 and 9
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hpi, the time in which ifnphi2 was regulated. ifnphi1 was upregulated from 12 hpi,
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similar to mx (a and b paralogues), which increased from 12 hours and reached a
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maximum at 24 hours. rarres3 showed a biphasic response, with higher expression at 9
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and 24 hpi. The recently characterized ifit gene family in zebrafish (41, 42) was also
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present in the response, highlighting the response of ifit17a, which reached a 20-fold
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change.
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SVCV positive cells are not endothelial cells. To determine whether endothelial cells
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were the primary targets of SVCV in this in vivo model of infection we performed an
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immunohistochemistry using an antibody against SVCV (Fig. 4). Using Fli1a-GFP
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infected larvae, SVCV was clearly visible in areas surrounding vessels (Fig. 4A). The
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3D reconstruction and volume clipping of the Fli1a channel showed that the SVCV
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positive cells were not endothelial cells (Fig. 4B, C, D). We observed crossing and
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circulating SVCV-positive cells, suggesting that these cells might be leukocytes
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interacting with the endothelium.
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Our hypothesis was confirmed by performing L-plastin labelling (Fig. 5). L-
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plastin positive cells were observed surrounding vessels and many of these cells co-
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localized with SVCV. Again, the 3D reconstruction of the confocal images conferred
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information about the cells and their position relative to the vessels (Fig. 5A). In
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addition, we observed that SVCV-infected cells showed viral antigen positive signal
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inside and on their surface (Fig. 5A). The signal detected on the cell surface might be
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associated with viral assembly, antigen presentation by infected cells or both (6). We
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observed cells labeled with only L-plastin (interacting or not with the endothelium),
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double-positive cells (interacting or not with the endothelium) and cellular debris
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positive for one or both markers. Large amounts of cellular debris were observed,
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always with L-plastin-positive cells phagocytising SVCV-positive particles in the same
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area (Fig. 5B). This cellular debris might play a role in the first line of defense against
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SVCV, as observed with other pathogens (43). Furthermore, defective debris clearance
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has been associated with persistent inflammatory diseases (44).
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Cellular response to SVCV infection. The transendothelial migration of leukocytes
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during inflammation has been well characterized (45). In the present study, the role of
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leukocytes during infection was explored using the Mpx-GFP zebrafish transgenic line
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combined with L-plastin immunohistochemistry (Fig. 6). Using this system, we were
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able to differentiate between macrophages (L-plastin+, Mpx-) and neutrophils (L-
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plastin+, Mpx+). After 24 hours of infection, practically all macrophages were
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undetectable (Fig. 6A) and a statistically significant migration of neutrophils to the head
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was observed (Fig. 6A, 6B, 6C). The loss of leukocytes in the caudal hematopoietic
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region was also confirmed (Fig. 6B). Using markers for both cellular types, marco for
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macrophages and mpx for neutrophils, we could check by qPCR the dynamics of that
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leukocytes population (Fig. 6D). The analysis of marco gene expression suggested an
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initial decrease in the macrophage population between 3 and 6 hpi and then,
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macrophages remained stable until 18 hpi. Thereafter, the macrophage population
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markedly reduced to nearly 0 at 24 hpi (Fig. 6D). Regarding neutrophils, mpx revealed
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no differences in the number of cells, thus confirming the migration observed in
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confocal images of these cells during the infection. Complementary experiments using
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other transgenic lines possessing fluorescent macrophages (Mpeg-GFP), neutrophils
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(Mpx-GFP) or thrombocytes (Cd41-GFP) showed that only macrophages were SVCV
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positive at 18 hpi (data not shown). Moreover, given the importance of platelets and
307
coagulation system in hemorrhagic infections (46) we used the transgenic line with
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fluorescent thrombocytes (fish equivalent to mammalian platelets) to detect a possible
309
thrombocytopenia or coagulopathy after SVCV infection. As no differences were found
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in thrombocyte behavior or count between control and SVCV-infected larvae (data not
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shown), we decided to focus our attention in macrophages.
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To further confirm macrophage role as preferred target for this hemorrhagic
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virus, at least during the first hours of the infection, we took advantage of the
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morpholino-mediated gene knockdown technique. We injected the morpholino (MO)
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PU.1 at a concentration of 1 mM to prevent the formation of macrophages in embryos
316
(47). A comparison of the normal fish and macrophage-depleted fish in these
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experimental infections showed a delay in the mortality of morphants (Fig. 6E),
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although eventually the mortality reached 100% in both groups. The duration of the MO
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effect was analyzed at an equivalent time of 48 hpi in non-infected fish. Marco
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expression was completely inhibited, as demonstrated using qPCR analysis (Fig. 6E). In
321
contrast, the population of neutrophils was not affected by the morpholino, as
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demonstrated by the mpx gene expression after 48 hpi (Fig. 6E). The approximate 9-
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hour delay in the mortality between both groups, suggested that macrophage presence is
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crucial for SVCV pathogenesis during the first hours of infection. Therefore, we
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analyzed the transcription levels of the SVCV N gene at 9 hpi (Fig. 6E). At this time,
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fish without macrophages had lower viral transcription levels compared with normal
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fish. Macrophage depletion delays the kinetics of viral transcription but is not enough to
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prevent the death of morphant fish. This effect likely reflects the high virulence of the
329
infection. As an obligate intracellular pathogen, SVCV infects to survive and even if the
330
primary target is not present, the virus could still infect other cells. However, the
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efficiency of infection is reduced in the first hours, resulting in a different timing of
332
mortality. This, plus the fact that the only SVCV-positive cells detected at 18 hpi are
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macrophages, suggested that these cells could be the primary target of the virus.
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SVCV elicits an inflammatory response that induces macrophages pyroptosis and
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IL1β release. The fact that macrophages were primarily infected with SVCV prompted
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us to examine the “disappearance” of these cells and determine whether pyroptosis is
337
involved. Pyroptosis might play a role in pathogen clearance, leading to inflammatory
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pathology, which is detrimental to the host but positively affects the spread of the
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pathogen (3). This programmed cell death depends on inflammasome activation and
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consequent Caspase-1 action (48). In zebrafish, Caspa associates with ASC, an
341
inflammasome adaptor (49). Moreover, it has recently been demonstrated that this
342
caspase is able to cleave Il1 in zebrafish (50). Using qPCR, we characterized the
343
regulation of some important genes involved in the inflammatory response (Fig. 7A).
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Three typical pro-inflammatory cytokines, il1, tnf and il6, were regulated during
345
infection. Il1 was the first responder, and a clear biphasic profile was observed. The
346
initial up-regulation of cytokine expression might be associated with the recognition of
347
SVCV by TLRs, followed by a second up-regulation of cytokine with a consequent
348
inflammatory response (51). This inflammatory response also triggered the response of
349
Tnf and Il6. Tnf was induced from 12 to 24 hpi. In addition, il6 showed a subtle
350
expression peak at 18 hpi. Strikingly, the response of this cytokine was delayed and
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351
weak. Similar results were observed in the caspa time course. A complete inhibition of
352
asc was observed at 9 hpi, then, the expression of asc reached a maximum at 18 hpi
353
(Fig. 7A).
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Immunohistochemistry against Caspa and SVCV showed that the macrophages
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remaining at 20 hpi were positive for Caspa (Fig. 7B). These macrophages showed
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interactions with infected cells (Fig. 7B). In addition, Mpeg1-positive and Caspa-
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positive vesicles, presumably from macrophages, were observed in the same area near
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SVCV-positive cells. Consistently with previus experiments, the 3D reconstruction
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revealed that infected macrophages had external SVCV-positive signals (Fig. 7B).
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Significant differences were found in Caspa levels between control and infected fish
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(Fig 7C) showing the participation of that protein in the infection.
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Il1 regulation is essential for a proper acute inflammatory response to an
363
infectious challenge. The implication of Caspa in Il1 secretion prompted us to
364
investigate the presence of that pro-inflammatory cytokine in infected larvae. An
365
antibody against zebrafish Il1 in combination with the Mpeg1-GFP transgenic line
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allowed us the detection of this cytokine in macrophages (Fig. 7D). A 3D reconstruction
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of the confocal images was generated to amplify the details, and two types of Il1-
368
positive macrophages were observed in SVCV-infected larvae. The first type was
369
SVCV-positive macrophages (Fig. 7E). The second type of macrophages localized near
370
zones with SVCV signal, was Il1 positive but was not positive for the virus (Fig. 7F).
371
The difference between these two macrophages types was the amount of Il1 detected.
372
Non-permissive macrophages in areas with SVCV-positive signal contained a higher
373
amount of Il1. Furthermore, the Mpeg1 signal was lost in favor of Il1 signal, which
374
could lead to the release of this cytokine. Il1 was detected in the macrophages of non17
375
infected fish, but the signal in these resting macrophages was clearly different that
376
observed in stimulated cells (Fig. 8A). Macrophages from infected fish showed a higher
377
amount of Il1 and what could be the release of this cytokine by microvesicle shedding
378
(Fig. 8B) or directly through the cell membrane (Fig. 8C) were observed. To assess the
379
membrane disruption produced during pyroptosis we used a cell membrane-impermeant
380
dye, PI. This dye was used in vivo and the uptake of PI by macrophages during SVCV
381
infection was confirmed (Fig. 8D). Furthermore, TUNEL assay showed IL1β releasing
382
cells dying during infection (Fig 8E).
383
18
384
DISCUSSION
385
In the present work we obtained images of the infection at the single cell level
386
using a zebrafish model, identifying macrophages as the preferred target of SVCV. We
387
were able to demonstrate for the first time that pyroptosis and Il1β release were
388
involved in the viral-induced cell dead in a whole organism. The direct observation of
389
such processes in individual cells provided a deeper knowledge of the inflammatory
390
mechanisms implicated in this viral disease.
391
Hemorrhagic viral diseases are globally observed with important pathogens,
392
such as Dengue Virus or Hantaviruses (52). Although hemorrhages have been
393
associated with the infection of endothelial cells (1), other cellular types could be the
394
main targets for viral infection (4). The lack of adequate in vivo infection models has
395
limited the research on viral pathogenesis. In SVCV-infected zebrafish larvae we
396
observed a loss of fluorescence of the endothelial marker Fli1a. However, this was not
397
due to infection of endothelial cells or cytopathic effects, and vascular integrity
398
remained intact, at least in the tail. The fact that haemorrhages always occur in the head
399
of the larva suggests the possibility that these occur as a result of damage or
400
permeability regulation in a particular kind of vessel, as it is known that the control of
401
the permeability may be different in different vessel types (53). The dysregulation of the
402
vascular endothelium, observed as a loss of Fli1a fluorescence, could reflect strong
403
immune activation during infection. In addition, the response induced in the rest of the
404
organism should also be considered. The downregulation of important transcription
405
factors, such as Fli1a and Ets1a, in endothelial cells shows the importance of a response
406
of the organism as a whole. It has been shown that the downregulation of Fli1 in
407
response to LPS is mediated through TLRs (54). Endothelial cells express different
19
408
receptors, such as Tlr3, Rig-1 and Mda5, among others. Based on this fact and the
409
qPCR results obtained in the present study, the activation of endothelial cells through
410
the Tlr3 might the cause of the loss of Fli1a expression, thereby inducing loss of
411
circulation, hemorrhages and inhibition in the development of hematopoietic precursors.
412
Recent studies have shown a relationship between Fli1 and glycosphingolipids
413
metabolism (55). Thus, we could consider Fli1a downregulation as a mechanism for
414
protection against viral infection in endothelial cells. Although, similar to other
415
hemorrhagic viral infections, we cannot rule out the possibility that endothelial cells
416
could be infected during the final stages of infection (4). However, this situation would
417
not be decisive for the pathogenesis of the disease.
418
Altogether, the qPCR results provide interesting data regarding the dynamics of
419
the response. The viral transcription began to emerge from 9 hpi, time in which we
420
observed the first increase in il1 transcription and the strong downregulation of asc.
421
With regard to the complete inhibition of asc transcription observed at 9 hpi, we cannot
422
rule out the involvement of the virus. Pathogens have developed diverse strategies to
423
block some of the components of the inflammasome (56). It has been shown that ASC
424
transcription is downregulated during the infection of keratinocytes with some types of
425
human papillomavirus (57). The host might also control this response (58); thus, it is
426
likely that, in this case, the host could control macrophage loss through the inhibition of
427
asc transcription. However, the potential blockade by the virus to favor viral spread
428
cannot be excluded. Furthermore, the accumulation of Caspa as an unprocessed protein
429
(pro-caspase) into the cells (59) would explain the weak transcriptional regulation
430
observed for this gene during the infection.
20
431
Recent studies have demonstrated the importance of the inflammasome in Il1
432
generation upon infection with RNA viruses (60) and showed that a biphasic response
433
might be associated with the two signals necessary for Il1 release from macrophages
434
(61). If we take into account of the results obtained also at protein level, the second
435
upregulation observed at 12 hpi might be related with inflammasome activation.
436
Inflammasome activation could be induced directly by the virus or cellular debris, but
437
further investigation is necessary to confirm the type or types of inflammasomes
438
implicated in this process. Moreover, as occurs with Influenza virus (62), and based in
439
the obtained qPCR results, we can not exclude the involvement of Tlr7 in
440
inflammasome activation and IL1β release.
441
The zebrafish model facilitated the capture of images showing Il1 release from
442
single macrophages during SVCV infection. The shedding of microvesicles containing
443
fully processed Il1 from the plasma membrane represents a major secretory pathway
444
for the rapid release of this cytokine (63). We observed vesicles presumably derived
445
from macrophages in areas with cellular debris or SVCV-positive cells. However, we
446
also observed the possible direct release of Il1 through the cellular membrane. Rarres3,
447
more commonly known as an antiviral protein, has recently been associated with
448
increased microtubule stability in cells (64), and cells with more stable microtubules
449
displayed increased pyroptosis (65). The fact that the expression profile of rarres3
450
overlaps with that of Il1 should be addressed in future studies.
451
The transcriptional regulation of some of the genes associated with pyroptosis
452
and the complete disappearance of macrophages before the onset of mortality, being
453
positive for Caspa and Il1β, suggested that pyroptosis is involved in SVCV infection. In
454
recent years, there have been many advances in research concerning this type of cell
21
455
death, particularly with regard to bacteria (66). In the case of viruses, our understanding
456
remains a step behind. It is known that some viruses cause pyroptosis of macrophages in
457
vitro, but so far, it is less clear whether this process occurs in vivo (67, 68). Using the
458
zebrafish model, we have provided the first description of viral-induced pyroptosis in
459
the context of a whole organism. The fact that a hemorrhagic viral infection was able to
460
induce macrophage pyroptosis in zebrafish, in the context of whole larvae, promotes the
461
use of this model organism for studying host-pathogen interactions.
462
Future studies will be particularly important for an in deep study of the role of
463
the endothelium, the inflammasome and pyroptosis in defense against the infection.
464
Indeed, without losing sight of the possible effects that the virus might have on the
465
regulation of the host response, the results obtained in the present study could be the
466
starting point to understand the antiviral immune response as a whole. Furthermore, the
467
application of these techniques and studies to other pathogens will improve the current
468
knowledge of host-pathogen interactions and increase the potential for the discovery of
469
new therapeutic targets.
470
22
471
ACKNOWLEDGMENTS
472
This work was supported by the projects CSD2007-00002 “Aquagenomics” and
473
AGL2011-28921-C03 from the Spanish Ministerio de Economía e Innovación and
474
European structural funds (FEDER) / Ministerio de Ciencia e Innovación (CSIC08-1E-
475
102). Our laboratory is also funded by ITN 289209 “FISHFORPHARMA” (EU) and
476
project 201230E057 from the Agencia Estatal Consejo Superior de Investigaciones
477
Científicas (CSIC).
478
The authors would like to thank Rubén Chamorro for technical assistance,
479
Professor Paul Martin (University of Bristol) for the provision of the L-plastin antibody
480
and Dr. John Hansen (Western Fisheries Research Center) for the provision of the Il1
481
antibody. Mónica Varela received predoctoral grants from the JAE Program (CSIC and
482
European structural Funds).
483
23
484
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33
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FIGURE LEGENDS
701
Figure 1. Infection course and loss of endothelium fluorescence in infected larvae
702
(A) Kaplan-Meier survival curve after infection with 2 nl of a SVCV solution of 3x106
703
TCID50/ml (p<0.001). (B) Quantification of SVCV N gene transcripts as a measure of
704
the viral amount in the larvae over time. Heat-killed SVCV was injected in order to
705
separate immune response against the initial viral load. At 3 hpi significant differences
706
in the quantification of SVCV N gene transcripts were found between heat-killed SVCV
707
and SVCV infected larvae. The samples were obtained from infected larvae at different
708
times post-infection. Each sample (technical triplicates) was normalized to the 18S
709
gene. To avoid differences due to the larval development stage, the samples were
710
standardized with respect to an uninfected control of the same age. Three biological
711
replicates (of 10 larvae each) were used to calculate the means ± standard error of mean.
712
Significant differences between infected and non-infected larvae at the same time point
713
were displayed as *** (0.0001<p<0.001), ** (0.001<p<0.01) or * (0.01<p<0.05). qPCR
714
results are representative of 2 independent experiments. (C) The common macroscopic
715
symptoms observed at 24 hpi were hemorrhages and blood accumulation around the
716
eyes and head (arrows). See also Movie S1-S3 in supplemental material. (D). Zebrafish
717
larvae head confocal images (maximal projections from multiple z-stacks) of infected 3
718
dpf Tg(Fli1a-GFP) larvae immunostained with L-plastin antibody at 24 hpi (20x
719
magnification) showing the loss of GFP fluorescence in infected fish. (E) Confocal
720
images (maximal projections from multiple z-stacks) of infected 3 dpf Tg(Fli1a-GFP)
721
larvae immunostained with L-plastin antibody at 24 hpi (20x magnification) of vessels
722
in the yolk sac and trunk showing the loss of GFP fluorescence in infected fish. (F)
34
723
Higher magnification images are shown morphological changes in L-plastin-positive
724
cells in SVCV-infected fish.
725
Figure 2. Endothelium integrity is conserved 24 hours post-infection. (A)
726
Microangiography performed at 24 hpi injecting tetramethylrhodamine into the caudal
727
vein of 3 dpf infected and control larvae. Tetramethylrhodamine remained inside
728
vessels, even in infected fish without Fli1a-GFP fluorescence. Higher magnification
729
images are shown in the right panels. (B) DIC images showed the dorsal aorta
730
morphological integrity (arrows) in 24 hpi and control larvae. Note that blood cells were
731
visible inside dorsal aorta. (C) TUNEL assay and acridine orange staining were
732
performed in 24 hpi and control larvae to detect cells with DNA damage and necrotic
733
dead cells, respectively. Fli1a-GFP fluorescence is absent in infected fish, but TUNEL
734
positive cells were detected in the ventral part of the tail, which contains the caudal
735
hematopoietic tissue. No differences were found between control and SVCV-infected
736
larvae stained with acridine orange. (D) Fluorescent images of Fli1a-GFP of 3dpf
737
SVCV-infected and control larvae showing the loss of fluorescence in endothelial cells
738
after 22 hours of infection. Higher magnification images showed the intact morphology
739
of the dorsal aorta in infected larvae even when the fluorescence was lost (arrow head).
740
(E) The localization of L-plastin positive cells in caudal hematopoietic tissue is
741
consistent with that of TUNEL positive cells at 24 hours post-infection. TUNEL assay
742
was performed in 24 hours SVCV-infected and control larvae to detect cells with DNA
743
damage. L-plastin-positive cells are absent in infected fish, but TUNEL positive cells
744
are detected in the ventral part of the tail, which contains the caudal hematopoietic
745
tissue. (F) Expression qPCR measurements of vascular endothelium-related genes. The
746
samples were obtained from infected larvae at different times post-infection. Each
35
747
sample (technical triplicates) was normalized to the 18S gene. To avoid differences due
748
to the larval development stage, the samples were standardized with respect to an
749
uninfected control of the same age. Three biological replicates were used to calculate
750
the means ± standard error of mean. Significant differences between infected and non-
751
infected larvae at the same time point were displayed as *** (0.0001<p<0.001), **
752
(0.001<p<0.01) or * (0.01<p<0.05).
753
Figure 3. Viral detection and antiviral response measure over time. (A) Expression
754
qPCR measurements of TLRs and RLRs associated with viral detection. (B) Expression
755
qPCR measurements of antiviral response-related genes. Samples were obtained from
756
infected larvae at different times post-infection. Each sample (technical triplicates) was
757
normalized to the 18S gene. To avoid differences due to the larval development stage,
758
the samples were standardized with respect to an uninfected control of the same age.
759
Three biological replicates were used to calculate the means ± standard error of mean.
760
Significant differences were displayed as *** (0.0001<p<0.001), ** (0.001<p<0.01) or
761
* (0.01<p<0.05).
762
Figure 4. SVCV positive cells are distinct from endothelial cells. (A) Confocal
763
images (maximal projections from multiple z-stacks) of 3 dpf infected Tg(Fli1a-GFP)
764
larvae immunostained with SVCV antibody at 20 hpi (60x magnification). (B) (C) (D)
765
3D reconstructions of the different zones shown in A. Sequential steps of volume
766
clipping of the Fli1a channel are shown from left to right. We classified SVCV-positive
767
cells as circulating- (white arrows) and interacting- (white head arrows) cells. The axis
768
numbers correspond to μm.
769
Figure 5. SVCV positive cells are L-plastin positive and Fli1a negative. (A)
770
Confocal images 3D reconstruction of different scenes observed during the infection of
36
771
3 dpf larvae. Double positive cells are observed around vessels, interacting or not with
772
the endothelium. (B) Confocal images (maximal projections from multiple z-stacks) of
773
infected Tg(Fli1a-GFP) larvae double-immunostained with SVCV and L-plastin
774
antibodies at 20 hpi (60x magnification). Panels on the right show 3D reconstructions of
775
the areas identified in the central panels.
776
Figure 6. Neutrophils and macrophages respond to SVCV. (A) Confocal images
777
(maximal projections from multiple z-stacks) of infected 3 dpf Tg(Mpx-GFP) larvae
778
immunostained with L-plastin antibody 24 hpi (20x magnification). Head showing the
779
loss of L-plastin-positive cells in infected fish. MPX-positive cells increase in the same
780
area as a consequence of the migratory wave from the yolk sac population. (B) View of
781
yolk sac and tail of infected and control larvae. A yolk sac neutrophil population was
782
observed in the control larvae. Macrophages are missing in infected larvae. (C)
783
Neutrophil count in the head of control and 24 hours SVCV-infected larvae (n=4). (D)
784
qPCR results of the dynamics of macrophages and neutrophils population during SVCV
785
infection. (E) PU.1 MO was used to block macrophages development. Macrophages-
786
depleted fish showed a delay in mortality of 9 hours after SVCV infection at 2 dpf
787
compared to controls. The results are representative of 3 independent experiments of 2
788
or 3 biological replicates each. qPCR of marco and mpx facilitated an assessment of the
789
efficiency of PU.1 MO at 48 hpi. Regarding SVCV N gene, the morphants showed less
790
viral transcription during first hours of infection, suggesting that macrophages are the
791
main target of SVCV during the first hours of infection. Significant differences were
792
displayed as *** (0.0001<p<0.001), ** (0.001<p<0.01) or * (0.01<p<0.05).
793
Figure 7.SVCV elicits an inflammatory response and also macrophages pyroptosis.
794
(A) Expression qPCR measurements of some important inflammatory response genes.
37
795
Three biological replicates were used to calculate the means ± standard error of mean.
796
Significant differences between infected and non-infected larvae at the same time point
797
were displayed as *** (0.0001<p<0.001), ** (0.001<p<0.01) or * (0.01<p<0.05). (B)
798
Confocal images (maximal projections from multiple z-stacks) of 3 dpf infected
799
Tg(Mpeg1-GFP) larvae immunostained with SVCV and Caspa antibodies at 22 hpi (60x
800
magnification). The last macrophages in the larvae were Caspa positive. Some of these
801
cells were infected (boxed area) and the other cells were interacting with a group of
802
infected cells (arrowhead). (The arrow shows potential macrophage pyroptosis,
803
observed as visible membrane vesicle shedding. The last panel shows a 3D
804
reconstruction of the infected macrophage in the boxed area, with a viral antigen
805
positive area on its surface (arrowhead). (C) Resting macrophages shown Caspa signal
806
inside the cell. The level of Caspa was significantly less in resting macrophages than
807
that in the macrophages of infected larvae. (D) Confocal images (maximal projections
808
from multiple z-stacks) of infected Tg(Mpeg1-GFP) larvae immunostained with SVCV
809
and Il1 antibodies at 20 hpi (60x magnification). Regarding the proportion of Il1 and
810
SVCV fluorescence observed, we distinguished two types of macrophages. (E) 3D
811
reconstruction of infected macrophages in the boxed area is displayed. Il1 signal is
812
visible inside cells, as the SVCV signal. SVCV antigens are also visible on the cell
813
surface. (F) 3D reconstruction of non-infected macrophages positive for Il1 in boxed is
814
displayed. Mpeg1 fluorescence is lost in favor of the Il1 signal. Cellular debris positive
815
for SVCV is visible around these macrophages.
816
Figure 8. Interleukin 1 release is induced by SVCV infection. (A) Resting
817
macrophages shown Il1 signal inside the cell. The level of il1 was significantly less
818
in resting macrophages than that in the macrophages of infected larvae. (B) The
38
819
macrophages in 3 dpf infected larvae showed shedding of Il1-positive vesicles. (C)
820
Il1 also could be released directly through membrane pores in the macrophages of
821
infected larvae. (D) In vivo PI staining of infected larvae showing macrophages PI
822
uptake through membrane pores. (E) 3D reconstruction of macrophages in an SVCV
823
infected larvae positive for IL1β and TUNEL are displayed.
39