The inhibitory activities and antiviral mechanism of Viola philippica aqueous extracts against grouper iridovirus infection in vitro and in vivo.

Grouper iridovirus (GIV) is one of the most serious pathogens in mariculture and causes high mortality rates in cultured groupers; then, effective medicines for controlling GIV infections are urgently needed. Viola philippica is a well-known medicinal plant, and the application of V. philippica aqueous extracts against GIV infection was assessed by different methods in this study. The results showed that the working concentration of V. philippica aqueous extracts was 10 mg/ml. V. philippica aqueous extracts below 10 mg/ml have no significant cytotoxic effects on cell viability, while extracts over 15 mg/ml decreased cell viability and showed cytotoxic activity. V. philippica aqueous extracts had excellent inhibitory effects against GIV infection in vitro and in vivo. The possible antiviral mechanism of V. philippica was further analysed, which indicated that V. philippica did no damages to GIV particles, but it could disturb GIV binding, entry and replication in host cells. V. philippica had the best inhibitory effects against GIV during viral infection stage of binding and replication in host cells. Overall, the results suggest that appropriate concentration of V. philippica aqueous extracts has great antiviral effects, making it an interesting candidate for developing effective medicines for preventing and controlling GIV infection in farmed groupers.

In vitro antiviral efficacy of moroxydine hydrochloride and ribavirin against grass carp reovirus and giant salamander iridovirus.

Moroxydine hydrochloride (Mor) and ribavirin (Rib) have been reported to exhibit multi-antiviral activities against DNA and RNA viruses, but their antiviral activities and pharmacologies have seldom been studied in aquaculture. This paper has selected 3 aquatic viruses including a double-stranded RNA virus (grass carp reovirus, GCRV), a single-stranded RNA virus (spring viraemia of carp virus, SVCV) and a DNA virus (giant salamander iridovirus, GSIV) for antiviral testing. The results showed that Mor and Rib can effectively control the infection of GCRV and GSIV in respective host cells. Further study was undertaken to explore the antivirus efficiencies and pharmacological mechanisms of Mor and Rib on GCRV and GSIV in vitro. Briefly, compounds showed over 50% protective effects at 15.9 µg ml-1 except for the group of GSIV-infected epithelioma papulosum cyprinid (EPC) cells treated with Mor. Moreover, Mor and Rib blocked the virus-induced cytopathic effects and apoptosis in host cells to keep the normal cellular structure. The expression of VP1 (GCRV) and major capsid protein (MCP; GSIV) gene was also significantly inhibited in the virus-infected cells when treated with Mor and Rib. Cytotoxicity assay verified the 2 compounds had no toxic effects on grass carp ovary (GCO) cells and EPC cells at ≤96 µg ml-1. In conclusion, these results indicated that exposing GCRV-infected GCO cells and GSIV-infected EPC cells to Mor and Rib could elicit significant antiviral responses, and the 2 compounds have been shown to be promising agents for viral control in the aquaculture industry.

Palmitic Acid Promotes Virus Replication in Fish Cell by Modulating Autophagy Flux and TBK1-IRF3/7 Pathway.

Palmitic acid is the most common saturated fatty acid in animals, plants, and microorganisms. Studies highlighted that palmitic acid plays a significant role in diverse cellular processes and viral infections. Accumulation of palmitic acid was observed in fish cells (grouper spleen, GS) infected with Singapore grouper iridovirus (SGIV). The fluctuated content levels after viral infection suggested that palmitic acid was functional in virus-cell interactions. In order to investigate the roles of palmitic acid in SGIV infection, the effects of palmitic acid on SGIV induced cytopathic effect, expression levels of viral genes, viral proteins, as well as virus production were evaluated. The infection and replication of SGIV were increased after exogenous addition of palmitic acid but suppressed after knockdown of fatty acid synthase (FASN), of which the primary function was to catalyze palmitate synthesis. Besides, the promotion of virus replication was associated with the down-regulating of interferon-related molecules, and the reduction of IFN1 and ISRE promotor activities by palmitic acid. We also discovered that palmitic acid restricted TBK1, but not MDA5-induced interferon immune responses. On the other hand, palmitic acid decreased autophagy flux in GS cells via suppressing autophagic degradation, and subsequently enhanced viral replication. Together, our findings indicate that palmitic acid is not only a negative regulator of TBK1-IRF3/7 pathway, but also a suppressor of autophagic flux. Finally, palmitic acid promotes the replication of SGIV in fish cells.

Fish Cholesterol 25-Hydroxylase Inhibits Virus Replication via Regulating Interferon Immune Response or Affecting Virus Entry.

Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-induced gene that catalyzes the oxidation of cholesterol to 25-hydroxycholesterol (25HC), which exerts broad-spectrum antiviral function. To investigate the roles of fish CH25H in Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection, we cloned and characterized a CH25H homolog from orange-spotted grouper (Epinephelus coioides) (EcCH25H). EcCH25H encoded a 271-amino-acid polypeptide, with 86 and 59% homology with yellow croaker (Larimichthys crocea) and humans, respectively. EcCH25H contained a conserved fatty acid (FA) hydroxylase domain and an ERG3 domain. EcCH25H expression was induced by RGNNV or SGIV infection, lipopolysaccharide (LPS) or poly (I:C) treatment in vitro. Subcellular localization showed that EcCH25H and mutant EcCH25H-M were distributed in the cytoplasm and partly colocalized with the endoplasmic reticulum. SGIV and RGNNV replication was decreased by EcCH25H overexpression, which was reflected in the reduced severity of the cytopathic effect and a decrease in viral gene transcription, but replication of both viruses was increased by knockdown of EcCH25H. Besides, the antiviral activity was dependent on its enzymatic activity. Treatment with 25HC significantly inhibited replication of SGIV and RGNNV. EcCH25H overexpression positively regulated the IFN-related molecules and proinflammatory cytokines, and increased both IFN and ISRE promoter activities. Moreover, 25HC treatment significantly suppressed SGIV and RGNNV entry into host cells. The similar inhibitory effect on SGIV entry was observed in EcCH25H overexpression cells. Taken together, our findings demonstrated that EcCH25H inhibited SGIV and RGNNV infection by regulating IFN signaling molecules, and might also influence viral entry via an effect on cholesterol.

Identification of Inhibitory Compounds Against Singapore Grouper Iridovirus Infection by Cell Viability-Based Screening Assay and Droplet Digital PCR.

Singapore grouper iridovirus (SGIV) is one of the major causative agents of fish diseases and has caused significant economic losses in the aquaculture industry. There is currently no commercial vaccine or effective antiviral treatment against SGIV infection. Annually, an increasing number of small molecule compounds from various sources have been produced, and many are proved to be potential inhibitors against viruses. Here, a high-throughput in vitro cell viability-based screening assay was developed to identify antiviral compounds against SGIV using the luminescent-based CellTiter-Glo reagent in cultured grouper spleen cells by quantificational measurement of the cytopathic effects induced by SGIV infection. This assay was utilized to screen for potential SGIV inhibitors from five customized compounds which had been reported to be capable of inhibiting other viruses and 30 compounds isolated from various marine organisms, and three of them [ribavirin, harringtonine, and 2-hydroxytetradecanoic acid (2-HOM)] were identified to be effective on inhibiting SGIV infection, which was further confirmed with droplet digital PCR (ddPCR). In addition, the ddPCR results revealed that ribavirin and 2-HOM inhibited SGIV replication and entry in a dose-dependent manner, and harringtonine could reduce SGIV replication rather than entry at the working concentration without significant toxicity. These findings provided an easy and reliable cell viability-based screening assay to identify compounds with anti-SGIV effect and a way of studying the anti-SGIV mechanism of compounds.

Effects of atrazine and iridovirus infection on survival and life-history traits of the long-toed salamander (Ambystoma macrodactylum).

Environmental contaminants and emerging infectious diseases are implicated as factors contributing to global amphibian declines. However, few studies have tested the interaction of these factors. We exposed six-week-old, larval long-toed salamanders (Ambystoma macrodactylum) to Ambystoma tigrinum virus (ATV; 0 or 10(3.5) plaque-forming units/ml) and sublethal concentrations of atrazine (0, 1.84, 18.4, and 184 microg/L) in a 4 x 2 factorial design for 30 d. We tested the effects of atrazine and virus on mass and snout-vent length (SVL) at metamorphosis and larval period as well as on rates of mortality and viral infectivity. We confirmed ATV transmission to A. macrodactylum via polymerase chain reaction, but infection rates were lower than expected, consistent with the theory predicting lower pathogen transmission to nonnative hosts. Larvae exposed to both atrazine and ATV had lower levels of mortality and ATV infectivity compared to larvae exposed to virus alone, suggesting atrazine may compromise virus efficacy. The highest atrazine level (184 microg/L) accelerated metamorphosis and reduced mass and SVL at metamorphosis significantly relative to controls. Exposure to ATV also significantly reduced SVL at metamorphosis. The present study suggests moderate concentrations of atrazine may ameliorate effects of ATV on long-toed salamanders, whereas higher concentrations initiate metamorphosis at a smaller size, with potential negative consequences to fitness.

Giant seaperch iridovirus (GSIV) induces mitochondria-mediated cell death that is suppressed by bongkrekic acid and cycloheximide in a fish cell line.

Giant seaperch iridovirus (GSIV) induces cell death by an unknown mechanism. We postulated that this mechanism involves mitochondria-mediated cell death. Cell viability assays revealed a steady increase in dead grouper fin cells (GF-1) after GSIV infection, from 11% at 2 days post-infection (dpi) to 67% at 5 dpi. Annexin V/PI staining revealed GSIV infection induced apoptosis in a steadily increasing fraction of cells, from 4% at 1 dpi to 29% at 5 dpi. Furthermore, post-apoptotic necrosis was apparent at 4 and 5 dpi in the late replication stage. In the early replication stage, JC-1 dye revealed mitochondrial membrane potential (ΔΨm) loss in 42% of infected cells at 1 dpi, increasing to 98% at 3 dpi. Phosphatidylserine (PS) exposure and loss of ΔΨm from apoptosis/necrosis was attenuated by treatment with the adenine nucleotide translocase inhibitor bongkrekic acid (BKA) and the protein synthesis inhibitor cyclohexamide (CHX). These data suggest GSIV induces GF-1 apoptotic/necrotic cell death through pathways that require newly synthesized protein and involve the mitochondrial function.

Singapore Grouper Iridovirus ORF75R is a Scaffold Protein Essential for Viral Assembly.

Singapore Grouper Iridovirus (SGIV) is a member of nucleo cytoplasmic large DNA viruses (NCLDV). This paper reports the functional analysis of ORF75R, a major structural protein of SGIV. Immuno fluorescence studies showed that the protein was accumulated in the viral assembly site. Immunogold-labelling indicated that it was localized between the viral capsid shell and DNA core. Knockdown of ORF75R by morpholinos resulted in the reduction of coreshell thickness, the failure of DNA encapsidation, and the low yield of infectious particles. Comparative proteomics further identified the structural proteins affected by ORF75R knockdown. Two-dimensional gel electrophoresis combined with proteomics demonstrated that ORF75R was phosphorylated at multiple sites in SGIV-infected cell lysate and virions, but the vast majority of ORF75R in virions was the dephosphorylated isoform. A kinase assay showed that ORF75R could be phosphorylated in vitro by the SGIV structural protein ORF39L. Addition of ATP and Mg(2+) into purified virions prompted extensive phosphorylation of structural proteins and release of ORF75R from virions. These data suggest that ORF75R is a novel scaffold protein important for viral assembly and DNA encapsidation, but its phosphorylation facilitates virion disassembly. Compared to proteins from other viruses, we found that ORF75R shares common features with herpes simplex virus VP22.

Antiviral properties of hemocyanin isolated from shrimp Penaeus monodon.

Penaeid shrimp aquaculture has suffered from many diseases, especially from viral origin such as white spot syndrome virus (WSSV). In an attempt to obtain antiviral-relevant proteins, two peptides with molecular masses at 73 and 75kDa were isolated from shrimp Penaeus monodon using affinity chromatography coupled with the purified WSSV or a fish iridovirus (Singapore grouper iridovirus, SGIV), and identified as hemocyanin by mass spectrometry. The results, using fish viruses capable of cell culture, showed for the first time that the hemocyanin had non-specific antiviral properties and no cytotoxicity against host cells.

Temporal and differential gene expression of Singapore grouper iridovirus.

Singapore grouper iridovirus (SGIV), an iridovirus in the genus Ranavirus, is a major pathogen that results in significant economic losses in grouper aquaculture. To investigate further its infective mechanisms, for the first time, a viral DNA microarray was generated for the SGIV genome to measure the expression of its predicted open reading frames simultaneously in vitro. By using the viral DNA microarray, the temporal gene expression of SGIV was characterized and the DNA microarray data were consistent with the results of real-time RT-PCR studies. Furthermore, different-stage viral genes (i.e. immediate-early, early and late genes) of SGIV were uncovered by combining drug treatments and DNA microarray studies. These results should offer important insights into the replication and pathogenesis of iridoviruses.