A New Zebrafish Model to Measure Neuronal α-Synuclein Clearance In Vivo.

The accumulation and aggregation of α-synuclein (α-SYN) is a common characteristic of synucleinopathies, such as Parkinson's Disease (PD), Dementia with Lewy Bodies (DLB) or Multiple System Atrophy (MSA). Multiplications of the wildtype gene of α-SYN (SNCA) and most point mutations make α-SYN more aggregate-prone, and are associated with mitochondrial defects, trafficking obstruction, and impaired proteostasis, which contribute to elevated neuronal death. Here, we present new zebrafish models expressing either human wildtype (wt), or A53T mutant, α-SYN that recapitulate the above-mentioned hallmarks of synucleinopathies. The appropriate clearance of toxic α-SYN has been previously shown to play a key role in maintaining cell homeostasis and survival. However, the paucity of models to investigate α-SYN degradation in vivo limits our understanding of this process. Based on our recently described imaging method for measuring tau protein clearance in neurons in living zebrafish, we fused human SNCA to the photoconvertible protein Dendra2 which enabled analyses of wt and A53T α-SYN clearance kinetics in vivo. Moreover, these zebrafish models can be used to investigate the kinetics of α-SYN aggregation and to study the mechanisms, and potential new targets, controlling the clearance of both soluble and aggregated α-SYN.

Imaging of viral neuroinvasion in the zebrafish reveals that Sindbis and chikungunya viruses favour different entry routes.

Alphaviruses, such as chikungunya virus (CHIKV) and Sindbis virus (SINV), are vector-borne pathogens that cause acute illnesses in humans and are sometimes associated with neuropathies, especially in infants and elderly patients. Little is known about their mechanism of entry into the central nervous system (CNS), even for SINV, which has been used extensively as a model for viral encephalopathies. We previously established a CHIKV infection model in the optically transparent zebrafish larva; here we describe a new SINV infection model in this host. We imaged in vivo the onset and progression of the infection caused by intravenous SINV inoculation. Similar to that described for CHIKV, infection in the periphery was detected early and was transient, whereas CNS infection started at later time points and was persistent or progressive. We then tested the possible mechanisms of neuroinvasion by CHIKV and SINV. Neither virus relied on macrophage-mediated transport to access the CNS. CHIKV, but not SINV, always infects endothelial cells of the brain vasculature. By contrast, axonal transport was much more efficient with SINV than CHIKV, both from the periphery to the CNS and between neural tissues. Thus, the preferred mechanisms of neuroinvasion by these two related viruses are distinct, providing a powerful imaging-friendly system to compare mechanisms and prevention methods of encephalopathies.

Through the looking glass: witnessing host-virus interplay in zebrafish.

Host-pathogen interactions can be very complex at all scales; understanding organ- or organism-level events require in vivo approaches. Besides traditional host models such as mice, the zebrafish offers an attractive cocktail of optical accessibility and genetic tractability, blended with a vertebrate-type immunity, where innate responses can easily be separated from adaptive ones. Applied to viral infections, this model has revealed unexpected idiosyncrasies among organs, which we believe may apply to the human situation. We also argue that the dynamic analysis of virus spread and immune response in zebrafish make this model particularly well suited to the exploration of the concept of infection tolerance and resistance in relation to viral diseases.

Contrasted innate responses to two viruses in zebrafish: insights into the ancestral repertoire of vertebrate IFN-stimulated genes.

Ease of imaging and abundance of genetic tools make the zebrafish an attractive model host to understand host-pathogen interactions. However, basic knowledge regarding the identity of genes involved in antiviral immune responses is still lagging in this species. We conducted a microarray analysis of the larval zebrafish response to two models of RNA virus infections with very different outcomes. Chikungunya virus (CHIKV) induces a rapid and protective IFN response. Infection with infectious hematopoietic necrosis virus is lethal and is associated with a delayed and inefficient IFN response. A typical signature of IFN-stimulated genes (ISGs) was observed with both viruses, but was stronger for CHIKV. We further compared the zebrafish and human ISG repertoires and made a genomic and phylogenic characterization of the main gene families. We describe a core set of well-induced ISGs conserved across vertebrates, as well as multigenic families diversified independently in each taxon. The conservation of ISGs involved in antiviral signaling indicates conservation of the main feedback loops in these pathways. Whole-mount in situ hybridization of selected transcripts in infected larvae revealed a typical pattern of expression for ISGs in the liver, gut, and blood vessels with both viruses. We further show that some inflammatory genes were additionally induced through IFN-independent pathways by infectious hematopoietic necrosis virus and not by CHIKV. This study provides a useful reference set for the analysis of host-virus interactions in zebrafish and highlights the differences between protective and nonprotective antiviral innate responses.

The antiviral innate immune response in fish: evolution and conservation of the IFN system.

Innate immunity constitutes the first line of the host defense after pathogen invasion. Viruses trigger the expression of interferons (IFNs). These master antiviral cytokines induce in turn a large number of interferon-stimulated genes, which possess diverse effector and regulatory functions. The IFN system is conserved in all tetrapods as well as in fishes, but not in tunicates or in the lancelet, suggesting that it originated in early vertebrates. Viral diseases are an important concern of fish aquaculture, which is why fish viruses and antiviral responses have been studied mostly in species of commercial value, such as salmonids. More recently, there has been an interest in the use of more tractable model fish species, notably the zebrafish. Progress in genomics now makes it possible to get a relatively complete image of the genes involved in innate antiviral responses in fish. In this review, by comparing the IFN system between teleosts and mammals, we will focus on its evolution in vertebrates.

Real-time whole-body visualization of Chikungunya Virus infection and host interferon response in zebrafish.

Chikungunya Virus (CHIKV), a re-emerging arbovirus that may cause severe disease, constitutes an important public health problem. Herein we describe a novel CHIKV infection model in zebrafish, where viral spread was live-imaged in the whole body up to cellular resolution. Infected cells emerged in various organs in one principal wave with a median appearance time of ∼14 hours post infection. Timing of infected cell death was organ dependent, leading to a shift of CHIKV localization towards the brain. As in mammals, CHIKV infection triggered a strong type-I interferon (IFN) response, critical for survival. IFN was mainly expressed by neutrophils and hepatocytes. Cell type specific ablation experiments further demonstrated that neutrophils play a crucial, unexpected role in CHIKV containment. Altogether, our results show that the zebrafish represents a novel valuable model to dynamically visualize replication, pathogenesis and host responses to a human virus.

Sickle hemoglobin confers tolerance to Plasmodium infection.

Sickle human hemoglobin (Hb) confers a survival advantage to individuals living in endemic areas of malaria, the disease caused by Plasmodium infection. As demonstrated hereby, mice expressing sickle Hb do not succumb to experimental cerebral malaria (ECM). This protective effect is exerted irrespectively of parasite load, revealing that sickle Hb confers host tolerance to Plasmodium infection. Sickle Hb induces the expression of heme oxygenase-1 (HO-1) in hematopoietic cells, via a mechanism involving the transcription factor NF-E2-related factor 2 (Nrf2). Carbon monoxide (CO), a byproduct of heme catabolism by HO-1, prevents further accumulation of circulating free heme after Plasmodium infection, suppressing the pathogenesis of ECM. Moreover, sickle Hb inhibits activation and/or expansion of pathogenic CD8(+) T cells recognizing antigens expressed by Plasmodium, an immunoregulatory effect that does not involve Nrf2 and/or HO-1. Our findings provide insight into molecular mechanisms via which sickle Hb confers host tolerance to severe forms of malaria.

Whole-body analysis of a viral infection: vascular endothelium is a primary target of infectious hematopoietic necrosis virus in zebrafish larvae.

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