Development of Anti-Nervous Necrosis Virus Agents with Structure- and Cell-based Assays
Date Issued
2015
Date
2015
Author(s)
Huang, Yi-Cheng
Abstract
Nervous necrosis virus (NNV) is a highly contagious pathogen, responsible for severe losses incurred in the aquaculture industry. Currently, no commercially available antivirals against the virulence observed during very early stages of fish larvae development. Therefore, to facilitate the discovery of antivirals against NNV, establishing tools for discovering anti-NNV agents is important. In this study, we developed a novel cell viability-based screening assay as well as a structure-based survey of viral RNA-dependent RNA polymerase (RdRp). The cell viability-based screening assay conditions were optimized and the robustness of the assay was confirmed by a Z’ factor value ranging from 0.7 to 0.94. After screening a library of 2,000 small molecule compounds, 43 compounds with a virus inhibition capacity of ≥55% were identified. A cytochrome P450 inhibitor, proadifen hydrochloride, was validated with an EC50 value of 6.48 μM and a CC50 value of 20.63 μM. This compound reduced viral RNA level by 99.68% in grouper fin-1 cells (GF-1) at 5 days post-infection. Surprisingly, we found that 18 of 43 compounds act as neurotransmitter agents. For structure-based survey of NNV RdRp, the three-dimensional structure of NNV RdRp was modeled using the SWISS-MODEL server. Structure-based alignment of the modeled NNV RdRp and foot-and-mouth disease virus RdRp crystal structure (in complex with ribavirin triphosphate) indicated that the conformation of the ligand-binding pockets were highly conserved. Since ribavirin was shown to inhibit the RdRp from various viruses, it was thus tested for its ability to inhibit NNV infection by a dose-dependent assay and viral RNA determination in GF-1 cells. Ribavirin inhibited the virus-induced CPE by 54.41% at a concentration of 25 μM, and the viral RNA levels of both RdRp and capsid protein decreased significantly at 48 hours post-infection. These results indicate that ribavirin may interact with the RdRp of NNV to inhibit viral infection in GF-1 cells. In summary, novel tools for discovering anti-NNV agents with structure and cell-based assays have been established. The inhibitors of NNV were also revealed. These results may not only be used as antiviral therapies, but also help us to further understand the interactions between viruses and host cells.
Subjects
betanodavirus
nervous necrosis virus
drug screening
RNA-dependent RNA polymerase
homology modeling
Grouper
Type
thesis