Zebrafish as a Vertebrate Model for Studying Nodavirus Infections

Abstract

Nervous necrosis virus (NNV) is a neurotropic pathogenic virus affecting a multitude of marine and freshwater fish species that has a high economic impact on aquaculture farms worldwide. Therefore, the development of new tools and strategies aimed at reducing the mortality caused by this virus is a pivotal need. Although zebrafish is not considered a natural host for NNV, the numerous experimental advantages of this species make zebrafish an attractive model for studying different aspects of the disease caused by NNV, viral encephalopathy and retinopathy (VER). In this work, we established the best way and age to infect zebrafish larvae with NNV, obtaining significant mortalities in 3-day-postfertilization larvae when the virus was inoculated directly into the brain or by intramuscular microinjection. As occurs in naturally susceptible fish species, we confirmed that after intramuscular injection the virus was able to migrate to the central nervous system (CNS). As expected, due to the severe damage that this virus causes to the CNS, alterations in the swimming behavior of the zebrafish larvae were also observed. Taking advantage of the existence of transgenic fluorescent zebrafish lines, we were able to track the migration of different innate immune cells, mainly neutrophils, to the site of infection with NNV via the brain. However, we did not observe colocalization between the viral particles and neutrophils. RNA-Seq analysis of NNV-infected and uninfected larvae at 1, 3 and 5 days postinfection (dpi) revealed a powerful modulation of the antiviral immune response, especially at 5 dpi. We found that this response was dominated by, though not restricted to, the type I interferon system, the major defence mechanism in the innate immune response against viral pathogens. Therefore, as zebrafish larvae are able to develop the main characteristic of NNV infection and respond with an efficient immune arsenal, we confirmed the suitability of zebrafish larvae for modelling VER disease and studying different aspects of NNV pathogenesis, immune response and screening of antiviral drugs.

Susceptibility and Permissivity of Zebrafish (Danio rerio) Larvae to Cypriniviruses

“…These include genes encoding the antiviral GTPase proteins such as mxa , mxb , mxc , and mxe , as well as rsad2 (or vig-1 , viperin ). This is consistent with previous observations in zebrafish larvae infected with NNV [ 78 ], Zebrafish Picornavirus (Zfpv) [ 19 ], and CyHV-3-infected adult zebrafish [ 38 ]. In terms of the cytokine response, genes encoding IFN regulatory factors irf7 and irf9 were also part of this main cluster.…”

“…Indeed, in this present study, the list of the 250 most significant DEGs at 2 dpi is dominated by typical ISGs ( Table S4 ). This ISG induction in the absence of IFN detection is similar to previous studies with WT zebrafish larvae infected with nervous necrosis virus (NNV) [ 78 ]. In both studies, it is likely that IFN upregulation occurred prior to the earliest sampling point.…”

“…Notably, zebrafish irf3 was also among the top 250 most significant DEGs ( Table S4 ), however as STRING returned no results for this gene, it was not included in the network in Figure 9 . In addition, genes encoding other important elements of the IFN response, stat1a , stat1b, stat2 , and augmentation and regulation of this response such as isg15 [ 30 ] were also featured in this cluster, consistent with zebrafish larvae responses to HHV-1 [ 77 ] and NNV [ 78 ].…”

“…However, few viruses are known to naturally infect zebrafish [ 1 , 19 ], thus it would be useful to determine if any extant uncharacterized members of the family Alloherpesviridae naturally infect zebrafish as a primary host, as it would open up new avenues of investigation with a valuable homologous herpesvirus-host model in zebrafish. It is also possible that this lack of mortality is related to the viral dose or even inoculation site, both of which can impact the severity of viral infections in zebrafish larvae, as exemplified elsewhere [ 77 , 78 ].…”

“…One of these is dominated by gene-sets related to programmed cell death, the other is dominated by PRR signalling, pathogen and inflammatory response gene-sets. Notably, enrichment of the RIG-I-like signalling pathway, the Toll-like receptor signalling pathway, and the Herpes simplex virus 1 gene-sets are consistent with zebrafish larvae response to NNV infection [ 78 ]. In Cluster-1, the KEGG Necroptosis pathway (DRE04217) is the most significant positively enriched gene-set, and joint most significantly enriched gene-set overall ( Tables S8 and S9 ).…”