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

Abstract

Nervous 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.

FIG 1

Quantification analysis of NNV genomic RNA in zebrafish larvae exposed to the virus. RNA was extracted from a pool of five larvae taken from each group at 24 h postinfection. NNV cDNA was identified and quantitated by real-time RT-PCR targeting two capsid gene fragments, an 81-bp fragment (primer set 1) and a 203-bp fragment (primer set 2). Each bar represents the mean ± SE of triplicate readings from pooled larvae, and the data are representative of those from three independent experiments. The NNV expression levels were normalized against the β-actin expression level. All control groups and the group infected with inactivated virus were found to be negative by the two primer sets. dpf, days postfertilization; 2dpf pierced, eggs were pierced with a needle before infection; 2dpf chor−, eggs were manually dechorionated before infection.

FIG 2

Survival curves for zebrafish larvae infected with NNV by bath immersion on days 2, 4, 6, and 8 postfertilization. The data are representative of those from three independent experiments (n = 60 for each age group). Mock-infected larvae of all age groups had survival rates of 95 to 100%. dpf, days postfertilization.

FIG 3

Survival curves for zebrafish larvae infected with NNV by bath immersion on day 4 postfertilization with live virus versus or inactivated virus. The data are representative of those from three independent experiments (n = 60 for each group).

FIG 4

Methacrylate-embedded sections of zebrafish larvae at 4 dpf stained with toluidine blue. (A to C) Control mock-infected larvae; (D to F) NNV-infected larvae showing marked neuropil vacuolation, as well as a relative paleness of neurons involving both the brain and retina. The most prominent injury appears to be in the photoreceptor layer (yellow arrows) of the retina, with apparent nearly total lysis of the photoreceptors. dpf, days postfertilization.

FIG 5

Zebrafish larvae infected with NNV on day 2 postfertilization. (A) Schematic presentation of dechorionated larvae and pierced eggs. The schematics show eggs containing larvae (I), pierced larvae (II), and dechorionated larvae (III). The gray background represents perivitelline fluid. (B) Survival curves. The groups of eggs described in the legend to panel A were infected with NNV by bath immersion, and their survival rate was recorded. The data are representative of those from three independent experiments (n = 60 for each age group).

FIG 6

Temporal expression levels of genes for TNF-α, Mx, and IL-1β following NNV infection at different days postfertilization. RNA was extracted from a pool of five larvae taken from each group at 24 h postinfection. cDNA was used to quantify innate immune gene expression by real-time RT-PCR. Each bar represents the mean ± SE of triplicate readings from three independent experiments. Gene expression levels were normalized against the β-actin expression level. dpf, days postfertilization; 2dpf pierced, eggs were pierced with a needle before infection; 2dpf Chor−, eggs were manually dechorionated before infection. The area marked with shading represents results for larvae that underwent piercing and dechorionation at 2 dpf. Statistically significant differences are marked with asterisks.