Setting out zebrafish (Danio rerio) as a model to study nervous necrosis virus-host interaction.

Viral nervous necrosis is responsible for important economic losses in aquaculture facilities. The causative agent is the nervous necrosis virus (NNV). Four NNV species have been described, although only RGNNV and SJNNV have been detected in the Mediterranean area. RGNNV- SJNNV reassortants have also been isolated from several species. In order to design strategies to improve fish resistance to NNV, in vivo studies in commercial and model species are required to study the mechanisms underlying fish susceptibility to viral isolates. The aim of this work was to set up zebrafish as model of NNV infection. To fulfil this aim, zebrafish susceptibility to three NNV isolates was determined, and viral replication and innate immune response were characterized. Three days post-fertilisation zebrafish larvae were infected by intracerebral injection with 107 TCID50/mL of SJ93Nag (SJNNV), Dl956 (RGNNV from seabass), and RG/SJ (from seabream). Larvae were daily monitored for 4 days to record clinical signs and mortality. At 1 and 4 days post-infection (dpi), 3 pools of 6 larvae were sampled for viral genome quantification. Innate immune response was also assessed. Transcriptional analyses were completed by in vivo 3D imaging approaches on a zebrafish transgenic line expressing GFP in neutrophils (Tg (mpx:GFP) to monitor neutrophils recruitment in brain. RGNNV was the most virulent isolate compared to SJNNV and RG/SJ. These observations were consistent with viral genome replication, as the highest number of viral genome copies was in RGNNV-infected larvae. The induction of immune-related genes and the recruitment of neutrophils in brain, was also higher in RGNNV-infected larvae. Therefore, further experiments can be designed in this successfully model to better understand the mechanisms underlying NNV virulence in its hosts. Acknowledgments: Projects PID2020-115954RB-100/AEI/10.13039/501100011033 (Spanish Government) and EU H2020 VBN_22_73 (VetBioNet project).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Macrophage-B Cell Interactions in the Inverted Porcine Lymph Node and Their Response to Porcine Reproductive and Respiratory Syndrome Virus

Swine lymph nodes (LN) present an inverted structure compared to mouse and human, with the afferent lymph diffusing from the center to the periphery. This structure, also observed in close and distant species such as dolphins, hippopotamus, rhinoceros, and elephants, is poorly described, nor are the LN macrophage populations and their relationship with B cell follicles. B cell maturation occurs mainly in LN B cell follicles with the help of LN macrophage populations endowed with different antigen delivery capacities. We identified three macrophage populations that we localized in the inverted LN spatial organization. This allowed us to ascribe porcine LN MΦ to their murine counterparts: subcapsular sinus MΦ, medullary cord MΦ and medullary sinus MΦ. We identified the different intra and extrafollicular stages of LN B cells maturation and explored the interaction of MΦ, drained antigen and follicular B cells. The porcine reproductive and respiratory syndrome virus (PRRSV) is a major porcine pathogen that infects tissue macrophages (MΦ). PRRSV is persistent in the secondary lymphoid tissues and induces a delay in neutralizing antibodies appearance. We observed PRRSV interaction with two LN MΦ populations, of which one interacts closely with centroblasts. We observed BCL6 up-regulation in centroblast upon PRRSV infection, leading to new hypothesis on PRRSV inhibition of B cell maturation. This seminal study of porcine LN will permit fruitful comparison with murine and human LN for a better understanding of normal and inverted LN development and functioning

New Look at RSV Infection: Tissue Clearing and 3D Imaging of the Entire Mouse Lung at Cellular Resolution

Background: Respiratory Syncytial Virus (RSV) is the major cause of severe acute respiratory tract illness in young children worldwide and a main pathogen for the elderly and immune-compromised people. In the absence of vaccines or effective treatments, a better characterization of the pathogenesis of RSV infection is required. To date, the pathophysiology of the disease and its diagnosis has mostly relied on chest X-ray and genome detection in nasopharyngeal swabs. The development of new imaging approaches is instrumental to further the description of RSV spread, virus–host interactions and related acute respiratory disease, at the level of the entire lung. Methods: By combining tissue clearing, 3D microscopy and image processing, we developed a novel visualization tool of RSV infection in undissected mouse lungs. Results: Whole tissue analysis allowed the identification of infected cell subtypes, based on both morphological traits and position within the cellular network. Furthermore, 3D imaging was also valuable to detect the cytoplasmic viral factories, also called inclusion bodies, a hallmark of RSV infection. Conclusions: Whole lung clearing and 3D deep imaging represents an unprecedented visualization method of infected lungs to allow insight into RSV pathophysiology and improve the 2D histology analyses

Technological developments and 3D imaging for the diagnosis of aquaculture fish diseases

Session 07. Challenges of fish nutrition for sustainable aquacultureInternational audienc

New reporter zebrafish line unveils heterogeneity among lymphatic endothelial cells during development

International audienceBackground: In zebrafish, lymphatic endothelial cells (LECs) originate from multiple/several distinct progenitor populations and generate organ-specific lymphatic vasculatures. Cell fate and tissue specificities were determined using a combination of genetically engineered transgenic lines in which the promoter of a LEC-specific gene drives expression of a fluorescent reporter protein. Results: We established a novel zebrafish transgenic line expressing eGFP under the control of part of the zebrafish batf3 promoter (Basic Leucine Zipper ATF-Like Transcription Factor 3). Spatiotemporal examination of Tg(batf3MIN:eGFP) transgenic fish revealed a typical lymphatic expression pattern, which does not perfectly recapitulate the expression pattern of existing LEC transgenic lines. eGFP+ cells constitute a heterogeneous endothelial cell population, which expressed LEC and/or blood endothelial cells (BEC) markers in different tissues. In addition, we characterize the renal eGFP+ cell as a population of interest to study kidney diseases and regeneration. Conclusion: Our Tg(batf3MIN:eGFP) reporter zebrafish line provides a useful system to study LEC populations, of which heterogeneity depends on origin of progenitors, tissue environment and physiological conditions. We further developed a novel fish-adapted tissue clearing method, which allows deep imaging and 3D-visualization of vascular and lymphatic networks in the whole organism

Deficiency in hereditary hemorrhagic telangiectasia-associated Endoglin elicits hypoxia-driven heart failure in zebrafish

ABSTRACT Hereditary hemorrhagic telangiectasia (HHT) is a rare genetic disease caused by mutations affecting components of bone morphogenetic protein (BMP)/transforming growth factor-β (TGF-β) signaling in endothelial cells. This disorder is characterized by arteriovenous malformations that are prone to rupture, and the ensuing hemorrhages are responsible for iron-deficiency anemia. Along with activin receptor-like kinase (ALK1), mutations in endoglin are associated with the vast majority of HHT cases. In this study, we characterized the zebrafish endoglin locus and demonstrated that it produces two phylogenetically conserved protein isoforms. Functional analysis of a CRISPR/Cas9 zebrafish endoglin mutant revealed that Endoglin deficiency is lethal during the course from juvenile stage to adulthood. Endoglin-deficient zebrafish develop cardiomegaly, resulting in heart failure and hypochromic anemia, which both stem from chronic hypoxia. endoglin mutant zebrafish display structural alterations of the developing gills and underlying vascular network that coincide with hypoxia. Finally, phenylhydrazine treatment demonstrated that lowering hematocrit/blood viscosity alleviates heart failure and enhances the survival of Endoglin-deficient fish. Overall, our data link Endoglin deficiency to heart failure and establish zebrafish as a valuable HHT model

A new zebrafish model of oro-intestinal pathogen colonization reveals a key role for adhesion in protection by probiotic bacteria.

International audienceThe beneficial contribution of commensal bacteria to host health and homeostasis led to the concept that exogenous non-pathogenic bacteria called probiotics could be used to limit disease caused by pathogens. However, despite recent progress using gnotobiotic mammal and invertebrate models, mechanisms underlying protection afforded by commensal and probiotic bacteria against pathogens remain poorly understood. Here we developed a zebrafish model of controlled co-infection in which germ-free zebrafish raised on axenic living protozoa enabled the study of interactions between host and commensal and pathogenic bacteria. We screened enteric fish pathogens and identified Edwardsiella ictaluri as a virulent strain inducing a strong inflammatory response and rapid mortality in zebrafish larvae infected by the natural oro-intestinal route. Using mortality induced by infection as a phenotypic read-out, we pre-colonized zebrafish larvae with 37 potential probiotic bacterial strains and screened for survival upon E. ictaluri infection. We identified 3 robustly protective strains, including Vibrio parahaemolyticus and 2 Escherichia coli strains. We showed that the observed protective effect of E. coli was not correlated with a reduced host inflammatory response, nor with the release of biocidal molecules by protective bacteria, but rather with the presence of specific adhesion factors such as F pili that promote the emergence of probiotic bacteria in zebrafish larvae. Our study therefore provides new insights into the molecular events underlying the probiotic effect and constitutes a potentially high-throughput in vivo approach to the study of the molecular basis of pathogen exclusion in a relevant model of vertebrate oro-intestinal infection

New Look at RSV Infection: Tissue Clearing and 3D Imaging of the Entire Mouse Lung at Cellular Resolution

International audienceThis article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC B

Exploring Zebrafish Larvae as a COVID-19 Model: Probable Abortive SARS-CoV-2 Replication in the Swim Bladder

International audienceAnimal models are essential to understanding COVID-19 pathophysiology and for preclinical assessment of drugs and other therapeutic or prophylactic interventions. We explored the small, cheap, and transparent zebrafish larva as a potential host for SARS-CoV-2. Bath exposure, as well as microinjection in the coelom, pericardium, brain ventricle, or bloodstream, resulted in a rapid decrease of SARS-CoV-2 RNA in wild-type larvae. However, when the virus was inoculated in the swim bladder, viral RNA stabilized after 24 h. By immunohistochemistry, epithelial cells containing SARS-CoV-2 nucleoprotein were observed in the swim bladder wall. Our data suggest an abortive infection of the swim bladder. In some animals, several variants of concern were also tested with no evidence of increased infectivity in our model. Low infectivity of SARS-CoV-2 in zebrafish larvae was not due to the host type I interferon response, as comparable viral loads were detected in type I interferon-deficient animals. A mosaic overexpression of human ACE2 was not sufficient to increase SARS-CoV-2 infectivity in zebrafish embryos or in fish cells in vitro . In conclusion, wild-type zebrafish larvae appear mostly non-permissive to SARS-CoV-2, except in the swim bladder, an aerial organ sharing similarities with the mammalian lung

Protective effect of selected strains against gnotobiotic zebrafish larva infection by <i>E. ictaluri</i>.

<p><b>A</b>. Four dpf-old freshly hatched axenic zebrafish larvae were kept germ-free or incubated with selected protective bacterial strains for 2 days prior to exposure to <i>E. ictaluri</i> at 6 dpf. Mortality was monitored daily after <i>E. ictaluri</i> infection. Control non-infected larvae are shown in gray. Asterisks indicate significant difference from <i>E. ictaluri</i>-infected population (***p<0.001). <b>B</b>. Inflammation marker expression in pretreated <i>E. ictaluri</i>-infected larvae. qRT-PCR was performed on 5 individual larvae per group using primers specific to <i>il1b</i>, <i>tnfa</i> and <i>il10</i> on RNA extracted at 3 dpi ( = 9 dpf) from germ-free or <i>E. coli</i> MG6155 F′ pre-colonized zebrafish larvae exposed to <i>E. ictaluri</i> at 3 days post infection. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002815#s2" target="_blank">Results</a> were normalized to mean expression in germ-free animals; mean ± SEM. Asterisk indicates significant difference between <i>E. ictaluri</i>-infected and uninfected populations (**p<0.01, ***p<0.001).</p