Abstract
BackgroundGrass carp (Ctenopharyngodon idella) hemorrhagic disease is caused by an acute infection with grass carp reovirus (GCRV). The frequent outbreaks of this disease have suppressed development of the grass carp farming industry. GCRV104, the representative strain of genotype III grass carp (Ctenopharyngodon idella) reovirus, belongs to the Spinareovirinae subfamily and serves as a model for studying the strain of GCRV which encodes an outer-fiber protein. There is no commercially available vaccine for this genotype of GCRV. Therefore, the discovery of new inhibitors for genotype III of GCRV will be clinically beneficial. In addition, the mechanism of GCRV with fiber entry into cells remains poorly understood.MethodsViral entry was determined by a combination of specific pharmacological inhibitors, transmission electron microscopy, and real-time quantitative PCR.ResultsOur results demonstrate that both GCRV-JX01 (genotype I) and GCRV104 (genotype III) of GCRV propagated in the grass carp kidney cell line (CIK) with a typical cytopathic effect (CPE). However, GCRV104 replicated slower than GCRV-JX01 in CIK cells. The titer of GCRV-JX01 was 1000 times higher than GCRV104 at 24 h post-infection. We reveal that ammonium chloride, dynasore, pistop2, chlorpromazine, and rottlerin inhibit viral entrance and infection, but not nystatin, methyl-β-cyclodextrin, IPA-3, amiloride, bafilomycin A1, nocodazole, and latrunculin B. Furthermore, GCRV104 and GCRV-JX01 infection of CIK cells depended on dynamin and the acidification of the endosome. This was evident by the significant inhibition following prophylactic treatment with the lysosomotropic drug ammonium chloride or dynasore.ConclusionsTaken together, our data have suggested that GCRV104 enters CIK cells through clathrin-mediated endocytosis in a pH-dependent manner. We also suggest that dynamin is critical for efficient viral entry. Additionally, the phosphatidylinositol 3-kinase inhibitor wortmannin and the protein kinase C inhibitor rottlerin block GCRV104 cell entry and replication.
Highlights
Grass carp (Ctenopharyngodon idella) hemorrhagic disease is caused by an acute infection with grass carp reovirus (GCRV)
According to phylogenetic relationship between GCRV isolates, Max L. et al [10, 11] have demonstrated that the isolates of GCRV can be divided into three genotypes, with representative isolates genotype I (GCRV-873, GCRV-JX01), genotype II (GCRV-HZ08, GCRV106), and genotype III (GCRV104)
We investigate candidate inhibitors for genotype III grass carp reovirus (GCRV104) entry and infection
Summary
Infection and entry kinetics of GCRV104 and GCRV JX01 The typical CPE of GCRV104 was observed at 5 days post-infection. The viral infection and entry in CIK cells treated with NH4Cl was significantly lower than that of the control group (P < 0.01) (Fig. 2b) This data indicates that NH4Cl inhibits GCRV104 infection and entry. Western blots determined that viral protein vp was reduced in GCRV104 infected cells treated with NH4Cl (Fig. 2c). These studies suggest that the entry of GCRV104 is dependent on a low pH. Western blot results indicate that viral proteins were reduced in GCRV104 infected dynasore-treated cells (Fig. 3c). CPZ and pistop at specific concentrations all decreased significantly the infectivity percentage of GCRV104 (P < 0.01) Both rottlerin and wortmannin reduced GCRV104 infection, compared to non-treated cells (Fig. 4b). The PI3K inhibitor wortmannin and the PKC inhibitor rottlerin block, at least partially, GCRV104 entry into host cells
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