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In teleosts, the immunoglobulin classes produced by B cells are IgM, IgD, and IgT/IgZ. IgT was initially described as an immunoglobulin specialized in mucosal responses; accumulating evidence, however, shows that it is also involved in systemic immune responses. Two types of IgT/IgZ (IgT1 and IgT2) were previously described in common carp, but their further characterization was hampered by the lack of specific tool. In the current study, we developed and validated polyclonal antibodies against carp IgT1 and IgT2 and used them in combination with well validated monoclonal antibody against carp IgM (WCI12), to study the distribution of IgM + , IgT1 + and IgT2 + B cells or their secreted immunoglobulins in various mucosal and systemic organs of carp. Finally, we also preliminary assessed the B cell response to infection with the blood-borne parasite Trypanoplasma borreli. Using these tools, we report on the distinct expression of soluble immunoglobulins in systemic and mucosal compartments. IgT1 and IgM were expressed in mucosal as well as systemic organs and responded to systemic parasitic infection, whereas IgT2 was preferentially expressed at mucosal sites and did not respond to systemic infections. By studying the distribution of B cells in different organs, compartmentalization of the three B cell subtypes was observed in gills and gut, whereas splenic B cells appeared as organized clusters around ellipsoids. Our results provide insights into the distribution and to some extent the function of B cells in carp, indicating that our newly developed tools are valuable for future studies aiming at the further characterization of immune responses of carp to infections and vaccination., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Eltijani, Embregts, Magadan, Wang, Brugman, Boudinot, Wiegertjes and Forlenza.)

The CRISPR/Cas9 system is widely used to manipulate viral genomes. Although Alphaherpesvirinae genomes are large and complicated to edit, in recent years several Pseudorabies virus (PRV) mutants have been successfully generated using the CRISPR/Cas9 system. However, the application of CRISPR/Cas9 editing on another member of alpha herpesviruses, bovine herpesvirus-1 (BHV-1), is rarely reported. This paper reports a rapid and straightforward approach to manipulating herpesviruses genome using CRISPR/Cas9. The recombinant plasmids contained the left and right arm of the thymidine kinase ( TK ) gene of PRV or of the glycoprotein I ( gI ) and glycoprotein E ( gE ) of BHV-1. Upon the cleavage of the TK or gIgE gene by Cas9 protein, this was replaced by the enhanced green fluorescence protein ( eGFP ) by homologous recombination. With this approach, we generated recombinant TK-/eGFP+ PRV and gIgE-/eGFP+ BHV-1 mutants and then proceeded to characterize their biological activities in vitro and in vivo. In conclusion, we showed that alpha herpesvirus, including PRV and BHV-1, can be rapidly edited using the CRISPR/Cas9 approach paving the way to the development of animal herpesvirus vaccines.

Although vaccines have been widely used for many years, they have failed to control H9N2 avian influenza virus (AIV) in the field in China. The high level of maternal-derived antibodies (MDAs) against H9N2 virus contributes to the H9N2 influenza vaccine failure in poultry. The study aimed to generate a new vaccine to overcome MDAs interference in H9N2 vaccination in chickens. We used turkey herpesvirus (HVT) as a vaccine vector to express H9 hemagglutinin (HA) proteins. The recombinant HVT expressing H9 HA proteins (rHVT-H9) was successfully generated and characterized in primary chicken embryonic fibroblasts (CEFs). Western blot and indirect immunofluorescence assay (IFA) showed that the rHVT-H9 consistently expressed HA proteins. In addition, the rHVT-H9 had similar growth kinetics to the parent HVT. Preliminary animal experiments showed that compared to the conventional inactivated whole virus (IWV) vaccine, the rHVT-H9 stimulated robust humoral immunity in chickens with passively transferred antibodies (PTAs) that were used to mimic MDAs. Transmission experiments showed that the rHVT-H9 induced both humoral and cellular immunity in chickens with PTAs. Furthermore, we used mathematical models to quantify the vaccine's efficacy in preventing the transmission of H9N2 AIV. The results showed that the rHVT-H9 reduced the virus shedding period and decreased the reproduction ratio (R) value in chickens with PTAs after homologous challenge. However, the vaccination in this trial did not yet bring R  < 1. In summary, we generated a new rHVT-H9 vaccine, which stimulated strong humoral and cellular immunity, reducing virus shedding and transmission of H9N2 AIV even in the presence of PTAs in chickens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pan, Liu, Niu, Huang, Yan, Teng, Li, Beerens, Forlenza, de Jong and Li.)

Dietary supplementation of fish with β-glucans has been commonly associated with immunomodulation and generally accepted as beneficial for fish health. However, to date the exact mechanisms of immunomodulation by β-glucan supplementation in fish have remained elusive. In mammals, a clear relation between high-fibre diets, such as those including β-glucans, and diet-induced immunomodulation via intestinal microbiota and associated metabolites has been observed. In this study, first we describe by 16S rRNA sequencing the active naive microbiota of common carp intestine. Based on the abundance of the genus Bacteroides , well known for their capacity to degrade and ferment carbohydrates, we hypothesize that common carp intestinal microbiota could ferment dietary β-glucans. Indeed, two different β-glucan preparations (curdlan and MacroGard ® ) were both fermented in vitro , albeit with distinct fermentation dynamics and distinct production of short-chain fatty acids (SCFA). Second, we describe the potential immunomodulatory effects of the three dominant SCFAs (acetate, butyrate, and propionate) on head kidney leukocytes, showing effects on both nitric oxide production and expression of several cytokines ( il-1b , il-6 , tnfα , and il-10 ) in vitro . Interestingly, we also observed a regulation of expression of several gpr40L genes, which were recently described as putative SCFA receptors. Third, we describe how a single in vivo oral gavage of carp with MacroGard ® modulated simultaneously, the expression of several pro-inflammatory genes ( il-1b , il-6 , tnfα ), type I IFN-associated genes ( tlr3.1 , mx3 ), and three specific gpr40L genes. The in vivo observations provide indirect support to our in vitro data and the possible role of SCFAs in β-glucan-induced immunomodulation. We discuss how β-glucan-induced immunomodulatory effects can be explained, at least in part, by fermentation of MacroGard ® by specific bacteria, part of the naive microbiota of common carp intestine, and how a subsequent production of SFCAs could possibly explain immunomodulation by β-glucan via SCFA receptors present on leukocytes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Petit, de Bruijn, Goldman, van den Brink, Pellikaan, Forlenza and Wiegertjes.)

A tightly regulated innate immune response to trypanosome infections is critical to strike a balance between parasite control and inflammation-associated pathology. In this study, we make use of the recently established Trypanosoma carassii infection model in larval zebrafish to study the early response of macrophages and neutrophils to trypanosome infections in vivo. We consistently identified high- and low-infected individuals and were able to simultaneously characterise their differential innate response. Not only did macrophage and neutrophil number and distribution differ between the two groups, but also macrophage morphology and activation state. Exclusive to high-infected zebrafish, was the occurrence of foamy macrophages characterised by a strong pro-inflammatory profile and potentially associated with an exacerbated immune response as well as susceptibility to the infection. To our knowledge, this is the first report of the occurrence of foamy macrophages during an extracellular trypanosome infection., Competing Interests: SJ, ED, FH, MN, GL, GW, MF No competing interests declared, (© 2021, Jacobs et al.)

Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs exert anti-viral functions due to their involvement in protein synthesis shut off and recruitment of innate immune signaling intermediates. The largest RNA viruses, coronaviruses, impose great threat to public safety and animal health; however, the significance of SGs in coronavirus infection is largely unknown. Infectious Bronchitis Virus (IBV) is the first identified coronavirus in 1930s and has been prevalent in poultry farm for many years. In this study, we provided evidence that IBV overcomes the host antiviral response by inhibiting SGs formation via the virus-encoded endoribonuclease nsp15. By immunofluorescence analysis, we observed that IBV infection not only did not trigger SGs formation in approximately 80% of the infected cells, but also impaired the formation of SGs triggered by heat shock, sodium arsenite, or NaCl stimuli. We further demonstrated that the intrinsic endoribonuclease activity of nsp15 was responsible for the interference of SGs formation. In fact, nsp15-defective recombinant IBV (rIBV-nsp15-H238A) greatly induced the formation of SGs, along with accumulation of dsRNA and activation of PKR, whereas wild type IBV failed to do so. Consequently, infection with rIBV-nsp15-H238A strongly triggered transcription of IFN-β which in turn greatly affected rIBV-nsp15-H238A replication. Further analysis showed that SGs function as an antiviral hub, as demonstrated by the attenuated IRF3-IFN response and increased production of IBV in SG-defective cells. Additional evidence includes the aggregation of pattern recognition receptors (PRRs) and signaling intermediates to the IBV-induced SGs. Collectively, our data demonstrate that the endoribonuclease nsp15 of IBV interferes with the formation of antiviral hub SGs by regulating the accumulation of viral dsRNA and by antagonizing the activation of PKR, eventually ensuring productive virus replication. We further demonstrated that nsp15s from PEDV, TGEV, SARS-CoV, and SARS-CoV-2 harbor the conserved function to interfere with the formation of chemically-induced SGs. Thus, we speculate that coronaviruses employ similar nsp15-mediated mechanisms to antagonize the host anti-viral SGs formation to ensure efficient virus replication., Competing Interests: The authors have declared that no competing interests exist.

Mammalian macrophages can adopt polarization states that, depending on the exact stimuli present in their extracellular environment, can lead to very different functions. Although these different polarization states have been shown primarily for macrophages of humans and mice, it is likely that polarized macrophages with corresponding phenotypes exist across mammals. Evidence of functional conservation in macrophages from teleost fish suggests that the same, or at least comparable polarization states should also be present in teleosts. However, corresponding transcriptional profiles of marker genes have not been reported thus far. In this study we confirm that macrophages from common carp can polarize into M1- and M2 phenotypes with conserved functions and corresponding transcriptional profiles compared to mammalian macrophages. Carp M1 macrophages show increased production of nitric oxide and a transcriptional profile with increased pro-inflammatory cytokines and mediators, including il6, il12 and saa. Carp M2 macrophages show increased arginase activity and a transcriptional profile with increased anti-inflammatory mediators, including cyr61, timp2b and tgm2b. Our RNA sequencing approach allowed us to list, in an unbiased manner, markers discriminating between M1 and M2 macrophages of teleost fish. We discuss the importance of our findings for the evaluation of immunostimulants for aquaculture and for the identification of gene targets to generate transgenic zebrafish for detailed studies on M1 and M2 macrophages. Above all, we discuss the striking degree of evolutionary conservation of macrophage polarization in a lower vertebrate.

Macrophages play important roles in conditions ranging from host immune defense to tissue regeneration and polarize their functional phenotype accordingly. Next to differences in the use of L-arginine and the production of different cytokines, inflammatory M1 macrophages and anti-inflammatory M2 macrophages are also metabolically distinct. In mammals, M1 macrophages show metabolic reprogramming toward glycolysis, while M2 macrophages rely on oxidative phosphorylation to generate energy. The presence of polarized functional immune phenotypes conserved from mammals to fish led us to hypothesize that a similar metabolic reprogramming in polarized macrophages exists in carp. We studied mitochondrial function of M1 and M2 carp macrophages under basal and stressed conditions to determine oxidative capacity by real-time measurements of oxygen consumption and glycolytic capacity by measuring lactate-based acidification. In M1 macrophages, we found increased nitric oxide production and irg1 expression in addition to altered oxidative phosphorylation and glycolysis. In M2 macrophages, we found increased arginase activity, and both oxidative phosphorylation and glycolysis were similar to control macrophages. These results indicate that M1 and M2 carp macrophages show distinct metabolic signatures and indicate that metabolic reprogramming may occur in carp M1 macrophages. This immunometabolic reprogramming likely supports the inflammatory phenotype of polarized macrophages in teleost fish such as carp, similar to what has been shown in mammals., (Copyright © 2020 Wentzel, Janssen, de Boer, van Veen, Forlenza and Wiegertjes.)

Aquafeed companies aim to provide solutions to the various challenges related to nutrition and health in aquaculture. Solutions to promote feed efficiency and growth, as well as improving the fish health or protect the fish gut from inflammation may include dietary additives such as prebiotics and probiotics. The general assumption is that feed additives can alter the fish microbiota which, in turn, interacts with the host immune system. However, the exact mechanisms by which feed influences host-microbe-immune interactions in fish still remain largely unexplored. Zebrafish rapidly have become a well-recognized animal model to study host-microbe-immune interactions because of the diverse set of research tools available for these small cyprinids. Genome editing technologies can create specific gene-deficient zebrafish that may contribute to our understanding of immune functions. Zebrafish larvae are optically transparent, which allows for in vivo imaging of specific (immune) cell populations in whole transgenic organisms. Germ-free individuals can be reared to study host-microbe interactions. Altogether, these unique zebrafish features may help shed light on the mechanisms by which feed influences host-microbe-immune interactions and ultimately fish health. In this review, we first describe the anatomy and function of the zebrafish gut: the main surface where feed influences host-microbe-immune interactions. Then, we further describe what is currently known about the molecular pathways that underlie this interaction in the zebrafish gut. Finally, we summarize and critically review most of the recent research on prebiotics and probiotics in relation to alterations of zebrafish microbiota and immune responses. We discuss the advantages and disadvantages of the zebrafish as an animal model for other fish species to study feed effects on host-microbe-immune interactions., (Copyright © 2020 López Nadal, Ikeda-Ohtsubo, Sipkema, Peggs, McGurk, Forlenza, Wiegertjes and Brugman.)

Background: Infectious disease outbreaks form major setbacks to aquaculture production and to further development of this important sector. Cyprinid herpes virus-3 (CyHV-3) is a dsDNA virus widely hampering production of common carp (Cyprinus carpio), one of the most farmed fish species worldwide. Genetically disease resistant strains are highly sought after as a sustainable solution to this problem. To study the genetic basis and cellular pathways underlying disease resistance, RNA-Seq was used to characterize transcriptional responses of susceptible and resistant fish at day 4 after CyHV-3 infection., Results: In susceptible fish, over four times more differentially expressed genes were up-regulated between day 0 and 4 compared to resistant fish. Susceptible and resistant fish responded distinctively to infection as only 55 (9%) of the up-regulated genes were shared by these two fish types. Susceptible fish elicited a typical anti-viral response, involving interferon and interferon responsive genes, earlier than resistant fish did. Furthermore, chemokine profiles indicated that the two fish types elicited different cellular immunity responses. A comparative phylogenetic approach assisted in chemokine copies annotation pointing to different orthologous copies common to bony-fishes and even carp-specific paralogs that were differentially regulated and contributed to the different response of these two fish types. Susceptible fish up-regulated more ccl19 chemokines, which attract T-cells and macrophages, the anti-viral role of which is established, whereas resistant fish up-regulated more cxcl8/il8 chemokines, which attract neutrophils, the antiviral role of which is unfamiliar., Conclusions: Taken together, by pointing out transcriptional differences between susceptible and resistant fish in response to CyHV-3 infection, this study unraveled possible genes and pathways that take part in disease resistance mechanisms in fish and thus, enhances our understanding of fish immunogenetics and supports the development of sustainable and safe aquaculture.

Trypanosomes are important disease agents of humans, livestock and cold-blooded species, including fish. The cellular morphology of trypanosomes is central to their motility, adaptation to the host's environments and pathogenesis. However, visualizing the behaviour of trypanosomes resident in a live vertebrate host has remained unexplored. In this study, we describe an infection model of zebrafish ( Danio rerio ) with Trypanosoma carassii . By combining high spatio-temporal resolution microscopy with the transparency of live zebrafish, we describe in detail the swimming behaviour of trypanosomes in blood and tissues of a vertebrate host. Besides the conventional tumbling and directional swimming, T. carassii can change direction through a 'whip-like' motion or by swimming backward. Further, the posterior end can act as an anchoring site in vivo. To our knowledge, this is the first report of a vertebrate infection model that allows detailed imaging of trypanosome swimming behaviour in vivo in a natural host environment., Competing Interests: ÉD, SJ, FH, HS, RP, MC, GW, MF No competing interests declared, (© 2019, Dóró et al.)

Immune-modulatory effects of β-glucans are generally considered beneficial to fish health. Despite the frequent application of β-glucans in aquaculture practice, the exact receptors and downstream signalling remains to be described for fish. In mammals, Dectin-1 is a member of the C-type lectin receptor (CLR) family and the best-described receptor for β-glucans. In fish genomes, no clear homologue of Dectin-1 could be identified so far. Yet, in previous studies we could activate carp macrophages with curdlan, considered a Dectin-1-specific β-(1,3)-glucan ligand in mammals. It was therefore proposed that immune-modulatory effects of β-glucan in carp macrophages could be triggered by a member of the CLR family activating the classical CLR signalling pathway, different from Dectin-1. In the current study, we used primary macrophages of common carp to examine immune modulation by β-glucans using transcriptome analysis of RNA isolated 6 h after stimulation with two different β-glucan preparations. Pathway analysis of differentially expressed genes (DEGs) showed that both β-glucans regulate a comparable signalling pathway typical of CLR activation. Carp genome analysis identified 239 genes encoding for proteins with at least one C-type Lectin Domains (CTLD). Narrowing the search for candidate β-glucan receptors, based on the presence of a conserved glucan-binding motif, identified 13 genes encoding a WxH sugar-binding motif in their CTLD. These genes, however, were not expressed in macrophages. Instead, among the β-glucan-stimulated DEGs, a total of six CTLD-encoding genes were significantly regulated, all of which were down-regulated in carp macrophages. Several candidates had a protein architecture similar to Dectin-1, therefore potential conservation of synteny of the mammalian Dectin-1 region was investigated by mining the zebrafish genome. Partial conservation of synteny with a region on the zebrafish chromosome 16 highlighted two genes as candidate β-glucan receptor. Altogether, the regulation of a gene expression profile typical of a signalling pathway associated with CLR activation and, the identification of several candidate β-glucan receptors, suggest that immune-modulatory effects of β-glucan in carp macrophages could be a result of signalling mediated by a member of the CLR family.

In February 2017, the "Programma Mattone Internazionale Salute" (ProMis), that is the Italian Program for Internationalization of Regional Health Systems of the Ministry of Health (MoH), presented the first version of its Position Paper on Health Tourism, which embeds a first shared approach to the recommendations expressed by the European Committee of Regions (CoR) on "Age-Friendly" tourism. The CoR stresses the importance of local and regional authorities in the coordination of multi-sectoral policies such as healthcare, social assistance, transport, urban planning and rural development in relation to the promotion of mobility, security, accessibility of services, including health care and social services. "Age-friendly" tourism is an example of an innovative tourist offer that strives to meet the health needs of the entire "traveling" population, with an integrated and cross-sector approach that involves various organizations operating in sectors such as healthcare, accessibility and transport. The aim of the workshop was to explore the interest of the stakeholders to participate in a systemic action in the field of "health" tourism, and to identify priority implementation areas that offer opportunities to take advantage of validated, innovative experiences that strengthen the accessibility to health and social services in regional, national and international contexts. This effort provides the opportunity to take advantage of aligning the European Structural and Investment Funds (ESIF) to the development of tourism, coherently with the needs and resources of local and regional health authorities.

A number of Seriola species are currently farmed or being investigated as future aquaculture species in countries around the world. However they face a number of issues and limitations which will need to be overcome to ensure future stability and growth, one of which are disease outbreaks. Despite this, very little has been done to understand the immune system of Seriola species and very few immune genes have been characterised. Antimicrobial peptides (AMP) are naturally occurring low molecular weight polypeptides that play a major role in an organism's immune system and act effectively as a first line of defence. This investigation isolates the full length cDNA sequences of two AMP's, piscidin and hepcidin from the yellowtail kingfish (Seriola lalandi). The full-length cDNA of the piscidin gene encodes a 65 amino acid prepropeptide, containing a 25-residue peptide, predicted to form an amphipathic helix-loop-helix structure. Phylogenetic analysis using fish piscidin sequences, showed that this AMP is only found in bony fish within the Acanthomorpha clade and that a possible three groups within the piscidin family exists, with S. lalandi belonging to a particular group. The full-length cDNA of the hepcidin gene encodes a 90 amino acid preprohepcidin, which contains a typical RX(R/K)R motif for cleavage of the mature peptide which comprises of eight conserved cysteine residues. Phylogenetic analysis of known vertebrate hepcidin antimicrobial peptide (HAMP) sequences, shows sequences from the Neoteleostei clade of bony fish form two very separate groups, HAMP1 and HAMP2, with the S. lalandi hepcidin gene grouped with the HAMP1 sequences. HAMP2 sequences are found to have multiple copies within fish and genome analysis showed very clearly that these two groups of genes are located on separate regions on the genome, with the multiple HAMP2 copies formed from tandem gene duplications. Lastly, using qPCR the expression of the S. lalandi piscidin gene within healthy fish was highest within, spleen and gills and lowest in liver, whereas hepcidin was highest in the liver with little or no expression in the spleen and gills., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Although spring viremia of carp virus (SVCV) can cause high mortalities in common carp, a commercial vaccine is not available for worldwide use. Here, we report a DNA vaccine based on the expression of the SVCV glycoprotein (G) which, when injected in the muscle even at a single low dose of 0.1 µg DNA/g of fish, confers up to 100% protection against a subsequent bath challenge with SVCV. Importantly, to best validate vaccine efficacy, we also optimized a reliable bath challenge model closely mimicking a natural infection, based on a prolonged exposure of carp to SVCV at 15°C. Using this optimized bath challenge, we showed a strong age-dependent susceptibility of carp to SVCV, with high susceptibility at young age (3 months) and a full resistance at 9 months. We visualized local expression of the G protein and associated early inflammatory response by immunohistochemistry and described changes in the gene expression of pro-inflammatory cytokines, chemokines, and antiviral genes in the muscle of vaccinated fish. Adaptive immune responses were investigated by analyzing neutralizing titers against SVCV in the serum of vaccinated fish and the in vitro proliferation capacity of peripheral SVCV-specific T cells. We show significantly higher serum neutralizing titers and the presence of SVCV-specific T cells in the blood of vaccinated fish, which proliferated upon stimulation with SVCV. Altogether, this is the first study reporting on a protective DNA vaccine against SVCV in carp and the first to provide a detailed characterization of local innate as well as systemic adaptive immune responses elicited upon DNA vaccination that suggest a role not only of B cells but also of T cells in the protection conferred by the SVCV-G DNA vaccine.

A continued programme of research is essential to overcome production bottlenecks in any aquacultured fish species. Since the introduction of genetic and molecular techniques, the quality of immune research undertaken in fish has greatly improved. Thousands of species specific cytokine genes have been discovered, which can be used to conduct more sensitive studies to understand how fish physiology is affected by aquaculture environments or disease. Newly available transcriptomic technologies, make it increasingly easier to study the immunogenetics of farmed species for which little data exists. This paper reviews how the application of transcriptomic procedures such as RNA Sequencing (RNA-Seq) can advance fish research. As a case study, we present some preliminary findings using RNA-Seq to identify cytokine related genes in Seriola lalandi. These will allow in-depth investigations to understand the immune responses of these fish in response to environmental change or disease and help in the development of therapeutic approaches., (Copyright © 2017. Published by Elsevier Ltd.)

Here, we report a transcriptome sequence of Trypanoplasma borreli isolated from its natural host, the common carp, Cyprinus carpio The transcriptome allows an analysis of abundant cell surface proteins and acts as a comparator for understanding the evolution and pathogenicity of other Kinetoplastida , including several that infect humans., (Copyright © 2017 Carrington et al.)

Both endotherms and ectotherms (e.g., fish) increase their body temperature to limit pathogen infection. Ectotherms do so by moving to warmer places, hence the term "behavioral fever." We studied the manifestation of behavioral fever in the common carp infected by cyprinid herpesvirus 3, a native carp pathogen. Carp maintained at 24°C died from the infection, whereas those housed in multi-chamber tanks encompassing a 24°C-32°C gradient migrated transiently to the warmest compartment and survived as a consequence. Behavioral fever manifested only at advanced stages of infection. Consistent with this, expression of CyHV-3 ORF12, encoding a soluble decoy receptor for TNF-α, delayed the manifestation of behavioral fever and promoted CyHV-3 replication in the context of a temperature gradient. Injection of anti-TNF-α neutralizing antibodies suppressed behavioral fever, and decreased fish survival in response to infection. This study provides a unique example of how viruses have evolved to alter host behavior to increase fitness., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)