Mortality Caused by Bath Exposure of Zebrafish (Danio rerio) Larvae to Nervous Necrosis Virus Is Limited to the Fourth Day Postfertilization

Abstract

cNervous necrosis virus (NNV) is a member of the Betanodavirus genus that causes fatal diseases in over 40 species of fish worldwide. Mortality among NNV-infected fish larvae is almost 100%. In order to elucidate the mechanisms responsible for the susceptibility of fish larvae to NNV, we exposed zebrafish larvae to NNV by bath immersion at 2, 4, 6, and 8 days postfertilization (dpf). Here, we demonstrate that developing zebrafish embryos are resistant to NNV at 2 dpf due to the protection afforded by the egg chorion and, to a lesser extent, by the perivitelline fluid. The zebrafish larvae succumbed to NNV infection during a narrow time window around the 4th dpf, while 6-and 8-day-old larvae were much less sensitive, with mortalities of 24% and 28%, respectively.A quaculture is the fastest increasing type of food production system in the world, with an annual growth rate of 9% since 1985 (1). The fish produced by aquaculture account for about 50% of the total amount of fish produced for human consumption (1). Nevertheless, the rapid development of aquaculture is associated with environmental costs, such as habitat degradation, diseases, and pollution (2). Viral nervous necrosis disease (VNND) is one of the most devastating threats to cultured marine fish worldwide and results in great economic loss. The disease is caused by the nervous necrosis virus (NNV), a member of the Betanodavirus genus. The virus is highly contagious and virulent to at least 40 marine and brackish water fish species, including groupers, sea bass, temperate basses, barramundi, mullet, sea bream, and flounder (3).NNV is a spherical, nonenveloped virus with a bipartite, positive, single-stranded RNA genome. The virus genome is composed of RNA1 (3,107 nucleotides [nt]) and RNA2 (1,421 nt), which encode the RNA-dependent RNA polymerase (RdRp) (4) and the coat protein (CP), respectively (5). A subgenomic transcript of the RNA1 segment (RNA3) encodes the nonstructural proteins B2 and B1 (6, 7). The B2 protein prevents host RNA interference-mediated cleavage (8). The B1 protein is expressed at the early stage of infection and exhibits an anti-necrotic cell death function (7).Mortality among NNV-infected larvae and juveniles is almost 100%, resulting in serious economic losses to producers of highvalue fish species. Mature fish are more resistant to VNND but are still capable of horizontally transferring the virus to mature fish and vertically to their offspring. The high level of susceptibility to NNV of fish larvae and juveniles, including zebrafish (9), and the recent report describing an NNV outbreak in zebrafish (10) led us to establish an NNV-zebrafish larva infection model to study hostpathogen interactions in the early stages of development. Zebrafish characteristics make them an attractive model for studying host-microbe interactions and immune system development. The optical transparency of zebrafish larvae, the availability of transgenic lines, their small size, and the rapid development of zebrafish embryos are some of their many advant...

Zebrafish as a Vertebrate Model for Studying Nodavirus Infections

“…( 23 ) were able to reproduce NNV infection in both adult zebrafish and larvae, and relevant mortalities were observed in larvae microinjected with the virus, which was subsequently confirmed by Morick et al. ( 24 ) after bath exposure. The susceptibility of zebrafish larvae to NNV creates an opportunity to easily screen anti-NNV compounds in an in vivo model, as was performed with the antiviral drug ribavirin ( 41 ).…”

“…Although no viruses, with some exceptions, are typically known to naturally infect zebrafish and cause massive mortality episodes ( 9 , 10 ), several viruses (including those infecting humans) have been studied using zebrafish as a model of infection. In the case of fish, the main diseases caused by viruses have been reproduced in zebrafish: rhabdoviruses such as spring viremia of carp virus (SVCV) ( 11 – 15 ), snakehead rhabdovirus (SHRV) ( 16 ), viral haemorrhagic septicemia virus (VHSV) ( 17 ), and infectious haematopoietic necrosis virus (IHNV) ( 7 ); birnaviruses such as infectious pancreatic necrosis virus (IPNV) ( 18 , 19 ); iridoviruses such as infectious spleen and kidney necrosis virus (ISKNV) ( 20 , 21 ) or European sheatfish virus (ESV) ( 22 ); and nodaviruses such as nervous necrosis virus (NNV) ( 23 , 24 ). Interestingly, two publications reported that zebrafish can be naturally infected by different nodaviruses ( 25 , 26 ); this can probably occur as a consequence of increases in temperature and crowding ( 27 ).…”

Betanodavirus ability to infect juvenile European sea bass, Dicentrarchus labrax, at different water salinity

“…; Morick et al . ) and other ornamental species (Furusawa et al . ), and anadromous species, such as Atlantic salmon, Salmo salar (Korsnes et al .…”

“…On the other hand, several studies have highlighted the ability of betanodavirus to induce clinical signs and mortality in experimentally infected freshwater species, for example zebrafish, Danio rerio (Lu et al 2008;Morick et al 2015) and other ornamental species (Furusawa et al 2007), and anadromous species, such as Atlantic salmon, Salmo salar (Korsnes et al 2005).…”

The Zebrafish Perivitelline Fluid Provides Maternally-Inherited Defensive Immunity

“…The survival rate of individuals exposed to the bacterial suspension in PVF was significantly higher than those exposed to the suspension of bacteria in PBS (positive control) and presented no significant difference with zebrafish embryos not exposed to the pathogen (negative control). A previous report [ 33 ] has suggested a possible function of PVF against viral infection. In that case, viral infection was tested by bathing 2 dpf embryos without their chorions, or with their chorions pierced, with virus.…”