Evaluation on the antiviral effect of a hydroxycoumarin against infectious hematopoietic necrosis virus infection in vitro and in vivo.

Infectious hematopoietic necrosis (IHN) caused by the viral pathogen infectious hematopoietic necrosis virus (IHNV) is a highly contagious disease of salmonid species, resulting in significant economic impact. The previous study showed a hydroxycoumarin derivative 7-[6-(2-methylimidazole) hexyloxy] coumarin (D5) significantly inhibited spring viraemia of carp virus (SVCV) infection, suggesting that D5 may be useful as a potential anti-IHNV agent. In this study, D5 at the concentration of up to 10 mg/L significantly inhibited IHNV replication in epithelioma papulosum cyprini (EPC) cells with a maximum inhibitory rate of >90%, maintained mitochondrial membrane potential (ΔΨm) levels, and decreased IHNV-induced apoptosis in virus-infected cells. As the consequence of protection on mitochondria, D5 enhanced antioxidant enzyme activities and decreased reactive oxygen species (ROS) to maintain the antioxidant-oxidant balance of IHNV-infected EPC cells. For in vivo study, D5 via intraperitoneal injection exhibited an anti-IHNV effect in the virus-infected fish by substantially enhancing the survival rate. Meanwhile, up-regulation of six interferon (IFN) related gene expressions demonstrated that D5 may activate IFN-related expressions for inhibiting IHNV replication during the early stage of viral infection, which is beneficial for the continuous antiviral action on controlling low viral loads in rainbow trout juvenile. Thus, D5 effective regulated IHNV-induced undesirable conditions to be an excellent potential therapeutic agent against IHNV infection.

Synthesis and antiviral activity of coumarin derivatives against infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) is a highly contagious disease of juvenile salmonid species. However, robust anti-IHNV drugs currently are extremely scarce. For the purpose of seeking out anti-IHNV drugs, here a total of 24 coumarin derivatives are designed, synthesized and evaluated for their anti-viral activities. By comparing the half maximal inhibitory concentrations (IC50) of the 12 screened candidate drugs in epithelioma papulosum cyprini (EPC) cells infected with IHNV, the imidazole coumarin derivative C4 is selected for additional validation studies, with an IC50 of 2.53 µM at 72 h on IHNV glycoprotein. Further experiments revealed that C4 could significantly inhibit apoptosis and cellular morphological damage induced by IHNV. On account of these findings, derivative C4 could be a viable way of controlling IHNV and considered as a promising lead compound for the development of commercial drugs.

Synthesis and antiviral activity of a new arctigenin derivative against IHNV in vitro and in vivo.

Viral diseases in aquaculture were challenging because there are few preventative measures and/or treatments. Our previous study indicated that imidazole arctigenin derivatives possessed antiviral activities against infectious hematopoietic necrosis virus (IHNV). Based on the structure-activity relationship in that study, a new imidazole arctigenin derivative, 4-(8-(2-ethylimidazole)octyloxy)-arctigenin (EOA), was designed, synthesized and its anti-IHNV activity was evaluated. By comparing inhibitory concentration at half-maximal activity (IC50), we found that EOA (IC50 = 0.56 mg/L) possessed a higher antiviral activity than those imidazole arctigenin derivatives in our previous study. Besides, EOA could significantly decrease cytopathic effect (CPE) and viral titer induced by IHNV in epithelioma papulosum cyprinid (EPC) cells. In addition, EOA significantly inhibited apoptosis induced by IHNV in EPC cells. Further data verified that EOA inhibited IHNV replication in rainbow trout, with reducing 32.0% mortality of IHNV-infected fish. The results suggested that EOA was more stable with a prolonged inhibitory half-life in the early stage of virus infection (1-4 days). Consistent with above results, EOA repressed IHNV glycoprotein gene expression in virus sensitive tissues (kidney and spleen) in the early stage of virus infection. Moreover, histopathological evaluation showed that tissues from the spleen and kidney of fish infected with IHNV exhibited pathological changes. But there were no lesions in any of the tissues from the control group and EOA-treaten group. In accordance with the histopathological assay, EOA could elicited anti-inflammation response in non-viral infected rainbow trout by down-regulating the expression of cytokine genes (IL-8, IL-12p40, and TNF-α). Altogether, EOA was expected to be a therapeutic agent against IHNV infection in the field of aquaculture.

Preparation and evaluation of microencapsulated delivery system of recombinant interferon alpha protein from rainbow trout.

Diseases caused by viruses pose a significant risk to the health of aquatic animals, for which there are presently no efficacious remedies. Interferon (IFN) serving as an antiviral agent, is frequently employed in clinical settings. Due to the unique living conditions of aquatic animals, traditional injection of interferon is cumbersome, time-consuming and labor-intensive. This study aimed to prepare IFN microcapsules through emulsion technique by using resistant starch (RS) and carboxymethyl chitosan (CMCS). Optimization was achieved using the Box-Behnken design (BBD) response surface technique, followed by the creation of microcapsules through emulsification. With RS at a concentration of 1.27 %, a water­oxygen ratio of 3.3:7.4, CaCl2 at 13.67 %, CMCS at 1.04 %, the rate of encapsulation can escalate to 80.92 %. Rainbow trout infected with Infectious hematopoietic necrosis virus (IHNV) and common carp infected with Spring vireemia (SVCV) exhibited a relative survival rate (RPS) of 65 % and 60 % after treated with IFN microcapsules, respectively. Moreover, the microcapsules effectively reduced the serum AST levels and enhanced the expression of IFNα, IRF3, ISG15, MX1, PKR and Viperin in IHNV-infected rainbow trout and SVCV-infected carp. In conclusion, this integrated IFN microcapsule showed potential as an antiviral agent for treatment of viral diseases in aquaculture.

Antiviral activity of casein and αs2 casein hydrolysates against the infectious haematopoietic necrosis virus, a rhabdovirus from salmonid fish.

Salmonid fish viruses, such as infectious haematopoietic necrosis virus (IHNV), are responsible for serious losses in the rainbow trout and salmon-farming industries, and they have been the subject of intense research in the field of aquaculture. Thus, the aim of this work is to study the antiviral effect of milk-derived proteins as bovine caseins or casein-derived peptides at different stages during the course of IHNV infection. The results indicate that the 3-h fraction of casein and α(S2) -casein hydrolysates reduced the yield of infectious IHNV in a dose-dependent manner and impaired the production of IHNV-specific antigens. Hydrolysates of total casein and α(S2) -casein target the initial and later stages of viral infection, as demonstrated by the reduction in the infective titre observed throughout multiple stages and cycles. In vivo, more than 50% protection was observed in the casein-treated fish, and the kidney sections exhibited none of the histopathological characteristics of IHNV infection. The active fractions from casein were identified, as well as one of the individual IHNV-inhibiting peptides. Further studies will be required to determine which other peptides possess this activity. These findings provide a basis for future investigations on the efficacy of these compounds in treating other viral diseases in farmed fish and to elucidate the underlying molecular mechanisms of action. However, the present results provide convincing evidence in support of a role for several milk casein fractions as suitable candidates to prevent and treat some fish viral infections.

Potential application of antiviral coumarin in aquaculture against IHNV infection by reducing viral adhesion to the epithelial cell surface.

Due to the lack of relevant therapies for infectious haematopoietic necrosis virus (IHNV) infection, the viral outbreak invariably causes serious economic losses in salmonid species. In this study, we evaluated the anti-IHNV effects of 7-(6-benzimidazole) coumarin (C10) and 4-phenyl-2-thioxo-1,2,3,4-tetrahydro-5H-chromeno[4,3-d]pyrimidin-5-one (S5) in vitro and in vivo. The results revealed that C10 at 12.5 mg/L and S5 at 25 mg/L significantly inhibited IHNV replication in epithelioma papulosum cyprini (EPC) cells with a maximum inhibitory rate >90%, showing that IHNV-induced cytopathic effect (CPE) was alleviated by C10 and S5. There are two complementary effects on antiviral mechanism: 1. C10 completely inhibited IHNV infectivity when the virus was preincubated with C10 at 12.5 mg/L, determining that C10 may have a negative impact on IHNV binding to the cell; 2. C10 also up-regulated the gene expression of extracellular proto type galectin-1 (Gal1-L2) and a chimera galectin-3 (Gal3-L1) of EPC cells to inhibit IHNV adhesion. For the in vivo study, injection and immersion of the coumarins enhanced the survival rate of rainbow trout (Oncorhynchus mykiss) juveniles by 25% (at least) at 12 dpi. IHNV loads in the kidney and spleen were also obviously decreased at 96 h, and thus we considered that they had a delaying effect on IHNV replication in vivo. Meanwhile, C10 with a high stability in aquacultural water in immersion suppressed IHNV horizontal transmission by decreasing the viral loads in recipient fish. Overall, our data suggest that there is a positive effect of C10 and S5 against IHNV infection in aquaculture, and C10 had the potential to be a broad-spectrum antiviral against fish rhabdoviruses.

Evaluation on the antiviral activity of ribavirin against infectious hematopoietic necrosis virus in epithelioma papulosum cyprini cells.

Infectious hematopoietic necrosis virus (IHNV) causes high mortality in several economically important salmonid fishes, but there is no approved therapy up to now. To address the urgent need for therapeutics to combat IHNV infection, we investigate the anti-IHNV activities of 14 common antiviral agents using epithelioma papulosum cyprini (EPC) cells in this study. Among these agents, ribavirin shows the highest inhibition on IHNV replication, with an inhibitory percentage of 99.88%. And the 72 h half maximal inhibitory concentrations (IC50) of ribavirin on IHNV glycoprotein is 0.40 mg/L. In addition, ribavirin significantly inhibits apoptosis and cellular morphological damage induced by IHNV. Mechanistically, ribavirin could damage the viral particle of IHNV. Moreover, ribavirin could be used for prevention of IHNV infection. Therefore, ribavirin is considered to develop as a promising agent to treat IHNV infection.

Synthesis of arctigenin derivatives against infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) is a common pathogen that causes severe disease and huge economic losses in the salmonid aquaculture industry. Herein, a series of arctigenin derivatives are synthesized to evaluate their antiviral activity against IHNV. The results indicate that the length of linker and imidazole substituent groups play an important role in decreasing IHNV replication. In this study, the arctigenin-imidazole hybrid derivative 15 with an eight carbon atoms length of the linker reduces IHNV replication with an IC50 value of 1.3 µM. In addition, derivative 15 significantly inhibits apoptosis and cellular morphological damage induced by IHNV. Mechanistically, derivative 15 can not damage the viral particle directly. While time-of-addition and viral binding assays reveal that derivative 15 mainly affect the early replication of IHNV but do not interfere with IHNV adsorption. Overall, derivative 15 could be considered to develop as a promising agent to treat IHNV infection.

Ursolic acid from Prunella vulgaris L. efficiently inhibits IHNV infection in vitro and in vivo.

Infectious hematopoietic necrosis virus (IHNV) is a fish viral pathogen that causes severe disease and huge economic losses in the salmonid aquaculture industry. However, anti-IHNV drugs currently are scarce. For the purpose of seeking out anti-IHNV drugs, the anti-IHNV activities of 32 medicinal plants were investigated by using epithelioma papulosum cyprini (EPC) cells. Among these plants, Prunella vulgaris L. (PVL) showed the strongest inhibition on IHNV replication with an inhibitory percentage of 99.3% at the concentration 100 mg/L. Further studies demonstrated that ursolic acid (UA), a major constituent of PVL, also showed a highly effective anti-IHNV activity. The half-maximal inhibitory concentration (IC50) at 72 h of UA on IHNV was 8.0 µM. Besides, UA could significantly decrease cytopathic effect (CPE) and the viral titer induced by IHNV in EPC cells. More importantly, UA also showed a strong anti-IHNV activity in vivo, as indicated by increasing the survival rate of rainbow trout and inhibiting viral gene expression. Intraperitoneal injection of UA increased the relative percentage of survival of rainbow trout by 18.9% and inhibited IHNV glycoprotein mRNA expression by > 90.0% in the spleen at the 1st-day post-infection. Altogether, UA was expected to be a therapeutic agent against IHNV infection in aquaculture.

Inactivated infectious haematopoietic necrosis virus (IHNV) vaccines.

The inactivation dynamics of infectious haematopoietic necrosis virus (IHNV) by b-propiolactone (BPL), binary ethylenimine (BEI), formaldehyde or heat and the antigenic and immunogenic properties of the inactivated vaccines were evaluated. Chemical treatment of IHNV with 2.7 mm BPL, 1.5 mm BEI or 50 mm formaldehyde abolished virus infectivity within 48 h whereas heat treatment at 50 or 100 degrees C rendered the virus innocuous within 30 min. The inactivated IHNV vaccines were recognized by rainbow trout, Oncorhynchus mykiss, IHNV-specific antibodies and were differentially recognized by antigenic site I or antigenic site II IHNV glycoprotein-specific neutralizing monoclonal antibodies. The BPL inactivated whole virus vaccine was highly efficacious in vaccinated rainbow trout challenged by waterborne exposure to IHNV 7, 28, 42 or 56 days (15 degrees C) after immunization. The formaldehyde inactivated whole virus vaccine was efficacious 7 or 11 days after vaccination of rainbow trout but performed inconsistently when tested at later time points. The other vaccines tested were not efficacious.

Salmonid fish viruses and cell interactions at early steps of the infective cycle.

A flow cytometric virus-binding assay that directly visualizes the binding and entry of infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) and virus haemorrhagic septicaemia virus (VHSV) to several cell lines was established. The highest efficiency of binding was shown by the BF-2 cell line and this was used to study, at the attachment level, the interactions of these cells with salmonid fish viruses in coinfections, and to further determine if the earliest stage of the viral growth cycle could explain the previously described loss of infectivity of IHNV when IPNV is present. Our results demonstrated that IPNV binds to around 88% of cells either in single or dual infections, whereas IHNV attachment always decreased in the presence of any of the other viruses. VHSV binding was not affected by IPNV, but coinfection with IHNV reduced the percentage of virus-binding cells, which suggests competition for viral receptors or co-receptors. Internalization of the adsorbed IHNV was not decreased by coinfection with IPNV, so the hypothetical competence could be restricted to the binding step. Treatment of the cells with antiviral agents, such as amantadine or chloroquine, did not affect the binding of IPNV and VHSV, but reduced IHNV binding by more than 30%. Tributylamine affected viral binding of the three viruses to different degrees and inhibited IPNV or IHNV entry in a large percentage of cells treated for 30 min. Tributylamine also inhibited IHNV cytopathic effects in a dose-dependent manner, decreasing the virus yield by 4 log of the 50% endpoint titre, at 10 mm concentration. IPNV was also inhibited, but at a lower level. The results of this study support the hypothesis that IHNV, in contrast to VHSV or IPNV, is less efficient at completing its growth cycle in cells with a simultaneous infection with IPNV. It can be affected at several stages of viral infection and is more sensitive to the action of antiviral compounds.

Structural characterization and antiviral activity of lentinan from Lentinus edodes mycelia against infectious hematopoietic necrosis virus.

A novel lentinan (LNT-I) was extracted from Lentinus edodes mycelia, and purified by an anion-exchange DEAE cellulose column and Sephadex G-200 gel. The structural characterization of LNT-I was determined by gas chromatography-mass spectrometry, high performance gel permeation chromatography, Fourier transform infrared spectrometry and 1D-nuclear magnetic resonance spectroscopy. The results showed that LNT-I was a ß-(1 → 3)-glucan backbone with -(1 → 6)-glucosyl side-branching units terminated by mannosyl and galactosyl residues, and its molecular weight was 3.79 × 105 Da. LNT-I consisted of glucose, mannose and galactose with the molar ratio of 19.26:1.20:1.00. LNT-I represented the prominent antiviral activity to IHNV at MOI of 0.05 and 0.10, respectively. Direct inactivation and the antiviral ability in pre-addition, co-addition and post-addition to IHNV (MOI of 0.05) were 62.34%, 39.60%, 53.63% and 82.38%, respectively under 100 µg/mL of LNT-I. Antiviral mechanisms of LNT-I mainly involved in the direct inactivation and the inhibition of viral replication. Moreover, LNT-I significantly down-regulated the expression level of TNF-α, IL-2 and IL-11, and up-modulated the expression levels of IFN-1 and IFN-γ after challenging with IHNV. The results indicated that the inhibitory effects of LNT-I on IHNV infection were possibly attributed to its regulation of the innate immune responses and specific immunity.

Z-FA-FMK demonstrates differential inhibition of aquatic orthoreovirus (PRV), aquareovirus (CSRV), and rhabdovirus (IHNV) replication.

Benzyloxycarbonyl-phenylalanyl-alanyl-fluoromethyl ketone (Z-FA-FMK) is a protease inhibitor that has been shown to strongly inhibit mammalian orthoreovirus replication. Here we explore the ability of Z-FA-FMK to inhibit three important yet genetically discrete aquatic fish viruses: chum salmon aquareovirus (CSRV), piscine orthoreovirus (PRV), and the rhabdovirus infectious hematopoietic necrosis virus (IHNV). Z-FA-FMK significantly attenuated CSRV in vitro transcription and infectious yield following low-dose (2-20µM) exposure, yet a relatively high dose (200µM) was required to completely block CSRV replication. For PRV and IHNV, no significant attenuation of in vitro viral transcription was observed following low-dose (2-20µM) exposure; and although high dose (200µM) exposure significantly attenuated both PRV and IHNV transcription, neither was completely inhibited. These transcriptional results were similarly reflected in IHNV infectious titre observed at 7days post exposure. PRV titre is currently undeterminable in vitro; however, in vivo intra-peritoneal injection of PRV into juvenile Atlantic salmon (Salmo salar) in conjunction with 1.5mg/kg Z-FA-FMK did not affect PRV replication as measured by blood associated viral transcripts at 14days post challenge. These results indicate that aquatic ortho- and aqua-reoviruses appear to possess resilience to Z-FA-FMK relative to mammalian orthoreoviruses and suggest that environmental parameters or alternative mechanisms for viral replication may affect the efficacy of Z-FA-FMK as an antireoviral compound. Further, as Z-FA-FMK has been shown to irreversibly inhibit cysteine proteases such as cathepsins B and L in vitro at concentrations of ≤100µM, continued replication of IHNV (and possibly PRV) at 200µM Z-FA-FMK suggests that replication of these viruses can occur in a cathepsin-independent manner whereas CSRV likely requires cathepsins or similar cysteine proteases for successful replication.

In vitro effects of recombinant zebrafish IFN on spring viremia of carp virus and infectious hematopoietic necrosis virus.

In order to evaluate the effects of fish recombinant interferon (rIFN) on fish pathogenic rhabdoviruses, the zebrafish (Danio rerio) IFN (DreIFN) allele B gene was cloned and expressed in Escherichia coli. In addition, the effects of recombinant DreIFN (rDreIFN) on spring viremia of carp virus (SVCV), infectious hematopoietic necrosis virus (IHNV), and vesicular stomatitis virus (VSV) were surveyed in fish and chicken cells. The mature peptide of DreIFN allele B gene encodes 163 amino acids. Residues 3 and 98 are a pair of cysteines that likely form an intrachain disulfide bridge. rDreIFN protein was detected as a band at 21.6 kDa by SDS-PAGE. The purified rDreIFN has anti-SVCV and anti-IHNV activity of 3 x 10(4) U/mg-10(7) U/mg. The results indicate that rDreIFN has higher activity against SVCV and IHNV on epithelioma papulosum cyprinid (EPC) than on grass carp (C. idellus) ovary (CO) cell lines and no activity against VSV on chick embryo fibroblasts (CEF).

Effects of Three Types of Inactivation Agents on the Antibody Response and Immune Protection of Inactivated IHNV Vaccine in Rainbow Trout.

Infectious hematopoietic necrosis virus (IHNV) infects salmonid fish, resulting in high mortality and serious economic losses to salmonid aquaculture. Therefore, an effective IHNV vaccine is urgently needed. To select an inactivation agent for the preparation of an effective IHNV vaccine, rainbow trout were immunized with mineral oil emulsions of IHNV vaccines inactivated by formaldehyde, binary ethylenimine (BEI), or ß-propiolactone (BPL). The fish were challenged 8 weeks after vaccination, and their IgM antibody response and relative percent survival (RPS) were evaluated. The results show that formaldehyde, BEI, and BPL abolished IHNV HLJ-09 infectivity within 24, 48, and 24 h at final concentrations of 0.2%, 0.02%, and 0.01%, respectively. The mean levels of specific IgM, both in serum and mucus (collected from the skin surface and gills), for the three immunized groups (from high to low) ranked as follows: the BPL group, BEI group, and formaldehyde group. From weeks 5 to 9, the mean log2 serum titers of IgM in the BPL group were significantly higher compared with those of the other groups (p < 0.05) during the 9 weeks of observation after vaccination (immunized at weeks 0 and6). Mucus OD490 values of the BPL group were significantly higher compared with those of the other groups (p < 0.05) when reaching their peak at weeks 5 and 8, but the difference between the formaldehyde and BEI groups was not significant (p > 0.05). The BPL-inactivated whole-virus vaccine had the greatest protective effect on the rainbow trout after challenge by an intraperitoneal injection of live IHNV, with an RPS rate of 91.67%, which was significantly higher compared with the BEI (83.33%) and formaldehyde (79.17%) groups. These results indicate that the BPL-inactivated IHNV oil-adjuvant vaccine was more effective than the formaldehyde- or BEI-inactivated vaccines. The results of this study provide an important foundation for further studies on inactivated IHNV vaccines.

Dextrans produced by lactic acid bacteria exhibit antiviral and immunomodulatory activity against salmonid viruses.

Viral infections in the aquaculture of salmonids can lead to high mortality and substantial economic losses. Thus, there is industrial interest in new molecules active against these viruses. Here we describe the production, purification, and the physicochemical and structural characterization of high molecular weight dextrans synthesized by Lactobacillus sakei MN1 and Leuconostoc mesenteroides RTF10. The purified dextrans, and commercial dextrans with molecular weights ranging from 10 to 2000kDa, were assayed in infected BF-2 and EPC fish cell-line monolayers for antiviral activity. Only T2000 and dextrans from MN1 and RTF10 had significant antiviral activity. This was similar to results obtained against infectious pancreatic necrosis virus. However the dextran from MN1 showed ten-fold higher activity against hematopoietic necrosis virus than T2000. In vivo assays using the MN1 polymer confirmed the in vitro results and revealed immunomodulatory activity. These results together with the high levels of dextran production (2gL(-1)) by Lb. sakei MN1, indicate the compounds potential utility as an antiviral agent in aquaculture.

Peculiar monomeric interferon gammas, IFNγrel 1 and IFNγrel 2, in ginbuna crucian carp.

The existence of fish-specific isoforms of interferon (IFN)γ, known as IFNγ-related (IFNγrel), has been reported in several fish species. However, comparisons with deduced amino acid sequences of known IFNγrels among several fish species have indicated significant differences at the C-terminus basic amino acid continuous sequences, which indicate the existence of multiple IFNγrel isoforms. Two distinct cDNAs, encoding two IFNγrels, ifngrel 1 and ifngrel 2, were cloned from ginbuna crucian carp (Carassius auratus langsdorfii). Recombinant IFNγrel 1 and IFNγrel 2 have shown high antiviral activities against the lethal crucian carp hematopoietic necrosis virus. Both ligands exhibit biological activity as monomers despite the fact that the functional conformation of IFNγ is a homodimer. Both interferons have a high degree of sequence similarity, but differ in the C-terminus region. In this region, IFNγrel 1 contains a functional nuclear localization sequence which induces the translocation of green fluorescent protein from the cytoplasm to the nucleus. IFNγrel 2 lacks this sequence. These results indicate that IFNγrel 1 and IFNγrel 2 are functional antiviral cytokines. These structurally related ligands play distinct antiviral roles through different intracellular translocation mechanisms. Thus, IFNγrels form a novel, distinct subtype included in type II IFNs. The cyprinid fish IFNγ subtype currently consists of four members, including two IFNγ isoforms and two distinct additional IFNγrel isoforms specific to the fish.

Antiviral activities of flavonoids isolated from the bark of Rhus verniciflua stokes against fish pathogenic viruses In Vitro.

An 80% methanolic extract of Rhus verniciflua Stokes bark showed significant anti-viral activity against fish pathogenic infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) in a cell-based assay measuring virus-induced cytopathic effect (CPE). Activity-guided fractionation and isolation for the 80% methanolic extract of R. verniciflua yielded the most active ethyl acetate fraction, and methyl gallate (1) and four flavonoids: fustin (2), fisetin (3), butin (4) and sulfuretin (5). Among them, fisetin (3) exhibited high antiviral activities against both IHNV and VHSV showing EC(50) values of 27.1 and 33.3 µM with selective indices (SI = CC(50)/EC(50)) more than 15, respectively. Fustin (2) and sulfuretin (5) displayed significant antiviral activities showing EC50 values of 91.2-197.3 µM against IHNV and VHSV. In addition, the antiviral activity of fisetin against IHNV and VHSV occurred up to 5 hr post-infection and was not associated with direct virucidal effects in a timed addition study using a plaque reduction assay. These results suggested that the bark of R. verniciflua and isolated flavonoids have significant anti-viral activity against IHNV and VHSV, and also have potential to be used as anti-viral therapeutics against fish viral diseases.

In vitro antiviral activity of red alga, Polysiphonia morrowii extract and its bromophenols against fish pathogenic infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus.

Our previous investigation revealed that 80% methanolic extract of the red alga Polysiphonia morrowii has significant antiviral activities against fish pathogenic viruses, infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV). The present study was conducted to identify compounds attributed for its antiviral activities and investigate their antiviral activities against IHNV and IPNV. Activity-guided fractionation for 80% methanolic extract of Polysiphonia morrowii using a cell-based assay measuring virus-induced cytopathic effect (CPE) on cells yielded a 90% methanolic fraction, which showed the highest antiviral activity against both viruses among fractions yielded from the extract. From the fraction, two bromophenols were isolated and identified as 3-bromo-4,5-dihydroxybenzyl methyl ether (1) and 3-bromo-4,5-dihydroxybenzaldehyde (2) based on spectroscopic analyses. For both compounds, the concentrations to inhibit 50% of flounder spleen cell (FSP cell) proliferation (CC(50)) and each viral replication (EC(50)) were measured. In the pretreatment test, 3-bromo-4,5-dihydroxybenzyl methyl ether (1) and 3-bromo-4,5-dihy-droxybenzaldehyde (2) exhibited significant antiviral activities showing selective index values (SI = CC(50)/EC(50)) of 20 to 42 against both IHNV and IPNV. In direct virucidal test, 3-bromo-4,5-dihydroxybenzyl methyl ether (1) showed significant antiviral activités against both viruses while 3-bromo-4,5-dihydroxybenzaldehyde (2) was significantly effective against only IHNV. Although antiviral efficacies of both compounds against IHNV and IPNV were lower than those of ribavirin used as a positive control, our findings suggested that the red alga Polysiphonia morrowii and isolated two bromophenols may have potential as a therapeutic agent against fish viral diseases.

A chlorophyll c2 analogue from the marine brown alga Eisenia bicyclis inactivates the infectious hematopoietic necrosis virus, a fish rhabdovirus.

We screened in vitro antiviral activity against a salmonid pathogenic virus, infectious hematopoietic necrosis virus (IHNV), from the extracts of a total of 342 species of marine algae collected from the Japanese coastline. The anti-IHNV activity was found primarily in MeOH extracts, and the extract from one marine brown alga in particular, Eisenia bicyclis, showed high anti-IHNV activity. The anti-IHNV compound was isolated and purified as MC15 from the E. bicyclis extract, and the chemical structure was determined by several spectrometric analyses. The antiviral compound was proved to be a chlorophyll c2 derivative lacking the metal ion Mg(2+). MC15 showed similar antiviral activity against other salmonid enveloped viruses such as Paralichthys olivaceus virus and Oncorhynchus masou virus, and stability against any pH and temperatures up to 100 degrees C. No cytotoxicity was observed at up to 5 microg/ml. The antiviral mechanism of MC15 appears to be direct inactivation of the viral particles. A time course study showed that the inactivation of IHNV was completed within 40 min when 200 PFU of IHNV was reacted with MC15 at 800 ng/ml.