Tilapia Lake Virus-Induced Neuroinflammation in Zebrafish: Microglia Activation and Sickness Behavior.

In mammals, the relationship between the immune system and behavior is widely studied. In fish, however, the knowledge concerning the brain immune response and behavioral changes during brain viral infection is very limited. To further investigate this subject, we used the model of tilapia lake virus (TiLV) infection of zebrafish (Danio rerio), which was previously developed in our laboratory. We demonstrated that TiLV persists in the brain of adult zebrafish for at least 90 days, even when the virus is not detectable in other peripheral organs. The virions were found in the whole brain. During TiLV infection, zebrafish displayed a clear sickness behavior: decreased locomotor activity, reduced food intake, and primarily localizes near the bottom zone of aquaria. Moreover, during swimming, individual fish exhibited also unusual spiral movement patterns. Gene expression study revealed that TiLV induces in the brain of adult fish strong antiviral and inflammatory response and upregulates expression of genes encoding microglia/macrophage markers. Finally, using zebrafish larvae, we showed that TiLV infection induces histopathological abnormalities in the brain and causes activation of the microglia which is manifested by changes in cell shape from a resting ramified state in mock-infected to a highly ameboid active state in TiLV-infected larvae. This is the first study presenting a comprehensive analysis of the brain immune response associated with microglia activation and subsequent sickness behavior during systemic viral infection in zebrafish.

Antiviral Actions of 25-Hydroxycholesterol in Fish Vary With the Virus-Host Combination.

Cholesterol is essential for building and maintaining cell membranes and is critical for several steps in the replication cycle of viruses, especially for enveloped viruses. In mammalian cells virus infections lead to the accumulation of the oxysterol 25-hydroxycholesterol (25HC), an antiviral factor, which is produced from cholesterol by the cholesterol 25 hydroxylase (CH25H). Antiviral responses based on CH25H are not well studied in fish. Therefore, in the present study putative genes encoding for CH25H were identified and amplified in common carp and rainbow trout cells and an HPLC-MS method was applied for determination of oxysterol concentrations in these cells under virus infection. Our results give some evidence that the activation of CH25H could be a part of the antiviral response against a broad spectrum of viruses infecting fish, in both common carp and rainbow trout cells in vitro. Quantification of oxysterols showed that fibroblastic cells are capable of producing 25HC and its metabolite 7α,25diHC. The oxysterol 25HC showed an antiviral activity by blocking the entry of cyprinid herpesvirus 3 (CyHV-3) into KFC cells, but not spring viremia of carp virus (SVCV) or common carp paramyxovirus (Para) in the same cells, or viral haemorrhagic septicaemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV) into RTG-2 cells. Despite the fact that the CH25H based antiviral response coincides with type I IFN responses, the stimulation of salmonid cells with recombinant type I IFN proteins from rainbow trout could not induce ch25h_b gene expression. This provided further evidence, that the CH25H-response is not type I IFN dependent. Interestingly, the susceptibility of CyHV-3 to 25HC is counteracted by a downregulation of the expression of the ch25h_b gene in carp fibroblasts during CyHV-3 infection. This shows a unique interplay between oxysterol based immune responses and immunomodulatory abilities of certain viruses.

Type I interferon-dependent response of zebrafish larvae during tilapia lake virus (TiLV) infection.

Tilapia lake virus (TiLV; genus: Tilapinevirus, family: Amnoonviridae) is a recently characterised enveloped virus with a linear, negative-sense single-stranded RNA genome, which causes high mortality in tilapia species. In the present study, we demonstrated that zebrafish (Danio rerio) larvae are susceptible to TiLV infection upon systemic injection. TiLV replicated in zebrafish larvae and caused their high mortality (of about 70%). Histopathological examination revealed that TiLV infection caused pathological abnormalities in zebrafish larvae that were well visible within the brain. Moreover, gene expression analysis revealed that TiLV infection induced up-regulation of the expression of the immune-related genes encoding pathogen recognition receptors involved in sensing of viral dsRNA (rig-I (ddx58), tlr3, tlr22), transcription factors (irf3, irf7), type I interferon (infϕ1), antiviral protein (mxa), and pro-inflammatory cytokine (il-1ß). We also demonstrated the protective role of the recombinant zebrafish IFNϕ1 on the survival of zebrafish larvae during TiLV infection. Our results show the importance of type I IFN response during TiLV infection in zebrafish larvae and demonstrate that zebrafish is a good model organism to study interactions between TiLV - a newly emerging in aquaculture virus, and fish host.

Cytosolic Sensors for Pathogenic Viral and Bacterial Nucleic Acids in Fish.

Recognition of the non-self signature of invading pathogens is a crucial step for the initiation of the innate immune mechanisms of the host. The host response to viral and bacterial infection involves sets of pattern recognition receptors (PRRs), which bind evolutionarily conserved pathogen structures, known as pathogen-associated molecular patterns (PAMPs). Recent advances in the identification of different types of PRRs in teleost fish revealed a number of cytosolic sensors for recognition of viral and bacterial nucleic acids. These are DExD/H-box RNA helicases including a group of well-characterized retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) and non-RLR DExD/H-box RNA helicases (e.g., DDX1, DDX3, DHX9, DDX21, DHX36 and DDX41) both involved in recognition of viral RNAs. Another group of PRRs includes cytosolic DNA sensors (CDSs), such as cGAS and LSm14A involved in recognition of viral and intracellular bacterial dsDNAs. Moreover, dsRNA-sensing protein kinase R (PKR), which has a role in antiviral immune responses in higher vertebrates, has been identified in fish. Additionally, fish possess a novel PKR-like protein kinase containing Z-DNA binding domain, known as PKZ. Here, we review the current knowledge concerning cytosolic sensors for recognition of viral and bacterial nucleic acids in teleosts.

Viral infection-induced changes in the expression profile of non-RLR DExD/H-box RNA helicases (DDX1, DDX3, DHX9, DDX21 and DHX36) in zebrafish and common carp.

In mammals, several non-RLR DExD/H-box RNA helicases are involve in sensing of viral nucleic acids and activation of antiviral immune response, however their role in the immune defense of fish is much less known. In this study, the expression profile of non-RLR DExD/H-box RNA helicase genes: ddx1, ddx3, dhx9, ddx21 and dhx36, was studied in zebrafish (Danio rerio) and common carp (Cyprinus carpio L.) during infection with two RNA viruses: spring viremia of carp virus (SVCV) and Chum salmon reovirus (CSV). Bioinformatic analysis of the amino acid sequences of the core helicase of DDX1, DDX3, DHX9, DDX21 and DHX36 in zebrafish and common carp revealed presence of all conserved motifs found amongst all other species, with the exception of common carp DHX9 which do not possess motif V. The transcripts of studied DExD/H-box RNA helicases were found in zebrafish ZF4 cell line as well as in all studied organs from zebrafish and common carp. The expression study demonstrated the up-regulation of the expression of selected non-RLR DExD/H-box RNA helicases during viral infections in ZF4 cell line (in vitro study) and in zebrafish and common carp organs (in vivo study). DDX1 was the only DExD/H-box RNA helicase which expression was repetitively up-regulated during in vivo infections with SVCV and CSV in zebrafish and SVCV in common carp. In ZF4 cells and kidney of common carp, viral infection-induced up-regulation of DExD/H-box RNA helicases preceded the up-regulation of type I IFN gene. Our results suggest that studied non-RLR DExD/H-box RNA helicases might be involved in antiviral immune response in fish.

Antiviral response of adult zebrafish (Danio rerio) during tilapia lake virus (TiLV) infection.

Tilapia lake virus (TiLV) is a novel enveloped orthomyxo-like virus with a genome of 10 segments of linear negative-sense single-stranded RNA. It causes massive mortality of wild and farmed tilapia species and because of its spread in Asia, Africa, South and North America, it is considered a threat to tilapia aquaculture. Here, we have evaluated the possible use of zebrafish (Danio rerio) to study immune response and host-pathogen interactions during an infection with TiLV. Adult zebrafish were infected with TiLV by intraperitoneal (i.p) injection or by cohabitation. Increased viral load was observed in liver, spleen and kidney of i.p. injected fish at 1, 3, 6, and 14 days post infection (dpi) but not in fish from the cohabitation group (only liver was tested). We also demonstrated that in spleen and kidney i.p. injection of TiLV induced up-regulation of the expression of the immune-related genes encoding pathogen recognition receptors involved in sensing of viral dsRNA (rig-I, tlr3, tlr22), transcription factors (irf3, irf7), type I interferon (infϕ1), antiviral protein (mxa), pro-inflammatory (il-1ß, tnf-α, il-8, ifnγ1-2) and anti-inflammatory (il-10) cytokines, CD4 markers (cd4-1, cd4-2), and IgM (igm). Moreover, tissue tropism of TiLV and histopathological changes were analyzed in selected organs of i.p. injected zebrafish. Our results indicate that zebrafish is a good model to study mechanisms of the TiLV infection and to follow antiviral responses.

Evaluation of zebrafish (Danio rerio) as an animal model for the viral infections of fish.

Zebrafish (Danio rerio) is a laboratory model organism used in different areas of biological research including studies of immune response and host-pathogen interactions. Thanks to many biological tools available, zebrafish becomes also an important model in aquaculture research since several fish viral infection models have been developed for zebrafish. Here, we have evaluated the possible use of zebrafish to study infections with fish viruses that have not yet been tested on this model organism. In vitro studies demonstrated that chum salmon reovirus (CSV; aquareovirus A) and two alloherpesviruses cyprinid herpesvirus 1 (CyHV-1) and cyprinid herpesvirus 3 (CyHV-3) are able to replicate in zebrafish cell lines ZF4 and SJD.1. Moreover, CSV induced a clear cytopathic effect and up-regulated the expression of antiviral genes vig-1 and mxa in both cell lines. In vivo studies demonstrated that both CSV and CyHV-3 induce up-regulation of vig-1 and mxa expression in kidney and spleen of adult zebrafish after infection by i.p. injection but not in larvae after infection by immersion. CyHV-3 is eliminated quickly from fish; therefore, virus clearing process could be evaluated, and in CSV-infected fish, a prolonged confrontation of the host with the pathogen could be studied.