1. Susceptibility of zebrafish larvae to Nervous necrosis virus
Danny Morick1, Yaakov Felig1, Rita Smirnov2, Adi Inbal3 and Moshe Kotler1
1. Department of Pathology and the Lautenberg Center for General and Tumor Immunology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel
2. Department of Fisheries and Aquaculture, Ministry of Agriculture &Rural Development, Nir David, Israel
3. Department of Medical Neurobiology, Institute for Medical Research – Israel-Canada, The Hebrew University of Jerusalem, Jerusalem, Israel
Introduction:
Nervous Necrosis Virus (NNV) is a Betanodavirus that causes mortal 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 larvae susceptibility to NNV we have established a NNV- Zebrafish larvae infection model. Zebrafish larvae were waterborne infected with NNV at the age of 2, 4, 6 and 8 days post fertilization (dpf) and larvae survival rate was recorded. Furthermore, to determine whether the egg chorion and/or the egg cytosol apply layers of defense against NNV infection, we mock or NNV infected dechorionated and pierced chorion 2dpf larvae.
Results:
Figure 2: For evaluating the role of the egg- chorion and the egg-cytosol in protecting the larva from NNV infection, eggs were manually dechorionated or pierced.
Figure 1: For evaluating most efficient infection protocol, 4 dpf larvae were infected with NNV in different environmental conditions and larvae survival was recorded for 11 days.
Figure 3:Epoxy-resin embedded semithin sections of larvae, stained with toluidine-blue. 4dpf larvea group showed marked neuropil rarefaction /vacuolation, as well as relative paleness of neurons, involving both brain and retina.
Figure 4: PCR amplification of a NNV 203-bp capsid fragment extracted from larvea at 2,4,6 and 8 dpf.
Survival curves of zebrafish larvae (N=60), waterborne infected with NNV
Figure 7: Live virus is necessary for infecting 4 dpf larvae.
Figure 6: The survival rate of 2dpf Pierced chorion and dechorionated embryos was reduced by 24% and 37% respectively.
Figure 5: Infection of larvae on the 2, 4, 6 and 8 dpf resulted in 5%, 100%, 24% and 28% mortality, respectively.
Conclusions:
In this study, we established a simple waterborne NNV- Zebrafish larvae infection model. Although larvae are infected by NNV during the first 8 dpf, the "window" of mortality caused by the virus is mostly restricted to day 4 post fertilization.
The egg-chorion and for a lesser extent, the egg cytosol, provides layers of defense against NNV infection of Zebrafish larvae at the age of 2 dpf.