Linking Virus Discovery to Immune Responses Visualized during Zebrafish Infections

Abstract

The discovery of new viruses currently outpaces our capacity for experimental examination of infection biology. To better couple virus discovery with immunology, we genetically modified zebrafish to visually report on virus infections. After generating a strain that expresses green fluorescent protein (GFP) under an interferon-stimulated gene promoter, we repeatedly observed transgenic larvae spontaneously expressing GFP days after hatching. RNA sequencing of GFP-positive zebrafish revealed a picornavirus distantly related to known viruses. We detected this virus in publicly available sequencing data from seemingly asymptomatic zebrafish in research institutes world-wide. These data revealed widespread tissue tropism and identified the clonal CG2 strain as notably vulnerable to picornavirus infection. Infection induces the expression of numerous antiviral immune defense genes in zebrafish, an effect also evident in public data. Our study describes a prevalent picornavirus that infects zebrafish, and provides new strategies for simultaneously discovering viruses and their impact on vertebrate hosts..

Tracking Mechanisms of Viral Dissemination In Vivo

“…A recent study reported the creation of a strain that expresses GFP under an interferon-stimulated gene promoter. Here, GFP expression can be used to track immune antiviral response in larvae and thereby provides an ideal platform to develop new strategies for discovering viruses while testing their impact on vertebrate models [ 39 ].

Can zebrafish become a key in vivo model for the study of the molecular mechanisms involved in viral dissemination?

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Dirty Fish Versus Squeaky Clean Mice: Dissecting Interspecies Differences Between Animal Models of Interferonopathy

“…While zebrafish facilities around the world undoubtedly take great care in optimizing water quality in their aquaria, there is some evidence that pathogens are present in water across a large number of centers ( 74 ). A recent study reported evidence of a novel picornavirus-like pathogen transmitted via the environment—leading to spontaneous activation of interferon responses in otherwise healthy animals.…”

“…Infection was associated with no overt phenotype, but rather was identified using an isg15 transgenic reporter line. Intriguingly, evidence of widespread viral infection was identified in RNAseq datasets from 92 facilities across the world ( 74 ). It would therefore seem that “asymptomatic” zebrafish infection may be relatively commonplace in zebrafish research and act as somewhat of a confounding—although not necessarily unhelpful—factor in studying immune responses in these animals.…”

“…If asymptomatic, low-virulence viral infection does in fact trigger interferonopathy in humans, this too may provoke the type I interferon response observed in zebrafish models. Indeed, upregulated transcription of isg15— an interferon-stimulated gene known to be involved in the zebrafish immune response and the very transgenic reporter line used to identify the novel picornavirus-like pathogen endemic to zebrafish facilities across the world—has been reported in samhd1 , adar and rnaset2 defective zebrafish models throughout development ( 23 , 38 , 74 ). After hatching (around 2 days post fertilization), zebrafish larvae may be particularly susceptible to viral infection of the brain due to lack of a functional blood brain barrier (BBB) ( 89 ).…”

“…The role of virus exposure in the zebrafish interferon response could be further dissected by exploiting the ex utero development of zebrafish embryos to raise animals in a sterile environment. Bleaching zebrafish eggs at 24 h post-fertilization has been shown to prevent productive viral infection throughout embryogenesis and is a strategy commonly used to raise embryos in a pathogen-free environment ( 74 , 92 ). If bleached zebrafish mutants were to show an improved inflammatory phenotype compared to their conventionally reared counterparts, this would suggest a role for viral infection in triggering type I interferon response.…”