α-Lipoic Acid Exerts Its Antiviral Effect against Viral Hemorrhagic Septicemia Virus (VHSV) by Promoting Upregulation of Antiviral Genes and Suppressing VHSV-Induced Oxidative Stress.

Viral hemorrhagic septicemia virus (VHSV), belonging to the genus Novirhabdovirus, Rhabdoviridae family, is a causative agent of high mortality in fish and has caused significant losses to the aquaculture industry. Currently, no effective vaccines, Food and Drug Administration-approved inhibitors, or other therapeutic intervention options are available against VHSV. α-Lipoic Acid (LA), a potent antioxidant, has been proposed to have antiviral effects against different viruses. In this study, LA (CC50 = 472.6 µmol/L) was repurposed to exhibit antiviral activity against VHSV. In fathead minnow cells, LA significantly increased the cell viability post-VHSV infection (EC50 = 42.7 µmol/L), and exerted a dose-dependent inhibitory effect on VHSV induced-plaque, cytopathic effects, and VHSV glycoprotein expression. The time-of-addition assay suggested that the antiviral activity of LA occurred at viral replication stage. Survival assay revealed that LA could significantly upregulated the survival rate of VHSV-infected largemouth bass in both co-injection (38.095% vs. 1.887%, P < 0.01) and post-injection manner (38.813% vs. 8.696%, P < 0.01) compared with the control group. Additional comparative transcriptome and qRT-PCR analysis revealed LA treatment upregulated the expression of several antiviral genes, such as IRF7, Viperin, and ISG15. Moreover, LA treatment reduced VHSV-induced reactive oxygen species production in addition to Nrf2 and SOD1 expression. Taken together, these data demonstrated that LA suppressed VHSV replication by inducing antiviral genes expression and reducing VHSV-induced oxidative stress. These results suggest a new direction in the development of potential antiviral candidate drugs against VHSV infection.

Virucidal effects of various agents-including protease-against koi herpesvirus and viral haemorrhagic septicaemia virus.

In a search for alternative, environmentally friendly and effective disinfecting agents, a commercially available protease-Neutrase® -was tested in this work for inactivation of koi herpesvirus (KHV) and of viral haemorrhagic septicaemia virus (VHSV). For comparison, the stability of these viral pathogens in similar configurations at various pH values and concentrations of peracetic acid or quicklime, typically used for disinfection, was tested. Therefore, virus suspensions were incubated with various concentrations of different agents for 24 hr and the titre of the remaining infectious particles was determined by virus titration. Furthermore, the treatment of both viruses, with the agents at concentrations that were previously appointed as effective, was also examined in the presence of solid material (quartz sand). All procedures investigated in this study, including the protease treatment, were able to reduce the titre of KHV and VHSV below the detection limit of the titration. Although further studies are necessary, this is the first report of the application of a protease for the inactivation of the selected fish pathogens, demonstrating the great potential of the latter for disinfection.

Efficacy of algal Ecklonia cava extract against viral hemorrhagic septicemia virus (VHSV).

The inhibition efficacy of an extract from Ecklonia cava (E. cava) was studied to determine whether the extract and compounds exhibited inhibitory activity against VHSV in the fathead minnow (FHM) cell line and following oral administration to the olive flounder. Based on its low toxicity and effective concentration, the E. cava extract (Ext) and compounds (eckol and phlorofucofuroeckol A) were selected for further analysis. In the plaque reduction assay, simultaneous co-exposure of VHSV to Ext, eckol and phlorofucofuroeckol A showed a higher level of inhibition than the pre- and post-exposure groups. The antiviral activity in the FHM cell line was time-dependent and increased with the exposure time with the virus and Ext or the compounds. In the in vivo experiments, different Ext concentrations were orally administered to the olive flounder. In trial I, the relative percent survival (RPS) following oral administration of 500 and 50 µg/g/day of Ext was 31.25% and 12.50%, respectively. In trial II, the RPS for 1000, 500 and 50 µg/g/day of Ext was 31.57%, 0% and 0%, respectively. In trial III, the RPS after 1 and 2 weeks (1000 µg/g/day) of exposure to Ext was 26.31% and 31.57%, respectively. Oral administration of Ext (1000 µg/g/day) significantly induced inflammatory cytokine responses (IL-1ß, IL-6 and IFN-γ) at 1 and 2 days post-oral administration (dpa). Additionally, IFN-α/ß (7-12 dpa), ISG15 (2, 7 and 10 dpa) and Mx (7-12 dpa) were significantly activated in the olive flounder. In conclusion, we demonstrated an inhibitory ability of the E. cava extract and compounds against VHSV in the FHM cell line. Moreover, oral administration of the E. cava extract to the olive flounder enhanced antiviral immune responses and the efficacy of protection against VHSV, resulting in an anti-viral status in the olive flounder.

Antiviral effects of extracts from Celosia cristata and Raphanus sativus roots against viral hemorrhagic septicemia virus.

The antiviral activity of an extract mixture from Celosia cristata and Raphanus sativus was tested against viral hemorrhagic septicemia virus (VHSV). Pretreatment of EPC cells with this extract up to 72 h before VHSV infection markedly reduced the virus titer, but it had no effect when added after virus inoculation. In olive flounder that received 5 µg of extract per fish, Mx expression peaked at 48 h after treatment. In contrast, ISG15 and TLR2 expression peaked at 72 h, and that of TLR7 peaked at 48 h, followed by a slight decrease at 72 h, indicating that the antiviral activity was mediated by induction of gene expression involved in the innate immune response.

shRNAs targeting either the glycoprotein or polymerase genes inhibit Viral haemorrhagic septicaemia virus replication in zebrafish ZF4 cells.

Viral haemorrhagic septicaemia virus (VHSV) represents an important disease of finfish. To explore the potential of shRNAs to combat this disease nucleotide sequences of either the VHSV glycoprotein (G) or polymerase (L) gene were targeted. To test their function, shRNAs were expressed in zebrafish epithelial ZF-4 cells utilizing the zebrafish U6-2 promoter. Five of the six shRNA molecules successfully reduced VHSV replication by between 2 and 4 logs in titre relative to an irrelevant control shRNA at all MOIs and also reduced viral CPE at the highest MOI. To ensure that observed reductions in viral titre were dependent on shRNA silencing, potential non-specific antiviral responses were assessed. Only the ineffective shRNA, which formed an improper hairpin when analysed in silico, induced an antiviral response as measured by induction of interferon (ifnphi1) and Mx (MxA) genes. These results represent an important preliminary step in the generation of transgenic zebrafish resistant to VHSV.

Effects of a broad-spectrum caspase inhibitor, Z-VAD(OMe)-FMK, on viral hemorrhagic septicemia virus (VHSV) infection-mediated apoptosis and viral replication.

In the development of inactivated or attenuated viral vaccines for cultured fish, viral titers harvested from the cultured cells would be the most important factor for the determination of vaccine's cost effectiveness. In this study, we hypothesized that the lengthening of cell survival time by the inhibition of apoptosis can lead to an increase of the final titer of viral hemorrhagic septicemia virus (VHSV). To test the hypothesis, we investigated the effects of a broad-spectrum caspase inhibitor, Z-VAD(OMe)-FMK, on VHSV infection-mediated apoptosis in Epithelioma papulosum cyprini (EPC) cells and on the VHSV titers. VHSV infection induced the DNA laddering in EPC cells, and the progression of DNA fragmentation was in proportion to the CPE extension. The progression of DNA fragmentation in EPC cells infected with VHSV was clearly inhibited by exposure to Z-VAD(OMe)-FMK, and the inhibition was intensified according to the increase of the inhibitor concentration. These results confirmed the previous reports that the death of host cells by VHSV infection is through apoptosis. Cells infected with a recombinant VHSV, rVHSV-ΔNV-eGFP, that was generated from our previous study by replacement of the NV gene ORF with the enhanced green fluorescent protein (eGFP) gene ORF, showed earlier and more distinct DNA fragmentations compared to the cells infected with wild-type VHSV, suggesting the inhibitory role of the NV protein in VHSV-mediated apoptosis that was previously reported. The final viral titers in the supernatant isolated from Z-VAD(OMe)-FMK treated cells after showing an extensive CPE were significantly higher than the viral titers from cells infected with virus alone, indicating that the delay of apoptosis by Z-VAD(OMe)-FMK extended the survival time of EPC cells, which lengthen the time for VHSV replication in the cells. In conclusion, Z-VAD(OMe)-FMK-mediated inhibition of apoptosis significantly increased the final titers of both wild-type VHSV and rVHSV-ΔNV-eGFP, indicating that apoptosis inhibition can be a way to get higher titers of VHSV.

Identification of regulators of the early stage of viral hemorrhagic septicemia virus infection during curcumin treatment.

The effect of curcumin pretreatment (15-240 µM) in fathead minnow cells infected with viral hemorrhagic septicemia virus (VHSV) was evaluated. Cell viability, apoptosis and viral copy number were analyzed using Cell Counting Kit-8 assay, Annexin V staining, and reverse transcription-PCR, respectively. Pretreatment with 120 µM curcumin showed an increase in viability (>90% of mock) of VHSV-infected cells and reduction in the copy number (0.2-log reduction in VHSV N gene expression), reactive oxygen species and apoptosis in the cells without cytotoxic effects. To understand the mechanisms underlaying the antiviral effects of curcumin pretreatment, a comparative proteomic analysis was performed in four samples (M, mock; C, curcumin-treated; V, VHSV-infected; and CV, curcumin-treated VHSV-infected) in triplicate. In total, 185 proteins were detected. The analysis showed that three proteins, including heat shock cognate 71 (HSC71), actin, alpha cardiac muscle (ACTC1) and elongation factor 1 (EEF1) were differentially expressed between V and CV samples. Network analysis performed by Ingenuity Pathways Analysis (IPA) showed that HSC71 was the primary protein interacting with fibronectin (FN) 1, actins (ACTB, ACTG, F-actin) and gelsolin (GSN) in both V and CV samples and thus is a strong target candidate for the protection from VHSV infection at the viral entry stage. Our proteomics data suggest that curcumin pretreatment inhibits entry of VHSV in cells by downregulating FN1 or upregulating F-actin. For both proteins, HSC71 acts as a binding protein that modulates their functions. Furthermore, consistent with the effect of a heat shock protein inhibitor (KNK437), curcumin downregulated HSC71 expression with increasing viability of VHSV-infected cells and inhibited VHSV replication, suggesting that the downregulation of HSC71 could be responsible for the antiviral activity of curcumin. In conclusion, this study indicates that the suppression of viral entry by rearrangement of the F-actin/G-actin ratio via downregulating HSC71 is a plausible mechanism by which curcumin pretreatment controls the early stages of VHSV infection.

Comparison of treatments to inactivate viral hemorrhagic septicemia virus (VHSV-IVb) in frozen baitfish.

Current US state and federal fish health regulations target the spread of viral hemorrhagic septicemia virus-IVb (VHSV-IVb) through movement restrictions of live fish; however, they largely ignore the potential for the virus to be spread through commercial distribution and use of frozen baitfish from VHSV-IVb-positive regions. Some state laws do require treatment of frozen baitfish to inactivate VHSV, and additional methods have been proposed, but few scientific studies have examined the efficacy of these treatments. In this study, bluegills Lepomis macrochirus were challenged with VHSV-IVb and frozen to represent standard industry methods, disinfected by various treatments, and tested for infectious VHSV-IVb using virus isolation. The virus was isolated from 70% of fish subjected to 3 freeze/thaw cycles. All other treatment methods were effective in inactivating the virus, including treatment with isopropyl alcohol, mineral oil, salt and borax, and dehydration. Dehydration followed by rehydration is rapid and effective, and therefore, seems to be the best option for inactivating VHSV-IVb present in frozen baitfish while maintaining their usefulness as bait.

Melittin-loaded immunoliposomes against viral surface proteins, a new approach to antiviral therapy.

In this study, melittin, a well-characterized pore-forming lytic amphiphilic peptide susceptible to be vehiculized in lipid membranes, has been utilized to study their antiviral properties. For this purpose, an assay based on melittin loaded-immunoliposomes previously described by our group was adapted to antiviral purposes by means of monoclonal antibodies targeting the surface G glycoprotein of the fish viral haemorrhagic septicemia rhabdovirus (VHSV). We also studied the antiviral action of these immunoliposomes in vitro and the results showed that they are capable of inhibiting the VHSV infectivity by 95.2% via direct inactivation of the virus. Furthermore, the inhibition of the infectivity when treatments were added at different times post-infection and the analysis of the infection foci sizes suggested altogether that they also act by reducing the VHSV spread in cell culture and by killing the infected cells which express the G glycoprotein in their plasmatic membranes.

Antiviral specificity of the Solea senegalensis Mx protein constitutively expressed in CHSE-214 cells.

Interferons play a key role in fish resistance to viral infections by inducing the expression of antiviral proteins, such as Mx. The aim of the present study was to test the antiviral activity of the Senegalese sole Mx protein (SsMx) against RNA and DNA viruses pathogenic to fish, i.e. the infectious pancreatic necrosis virus (IPNV, dsRNA), the viral haemorrhagic septicaemia virus (VHSV, ssRNA), and the European sheatfish virus (ESV, dsDNA), using a CHSE-214 cell clone expressing this antiviral protein. A strong inhibition of IPNV and VHSV replication was recorded in SsMx-expressing cells, as has been shown by the virus yield reduction and the decrease in the synthesis of the viral RNA encoding the polyprotein (for IPNV) and the nucleoprotein (for VHSV). The titres of these viruses replicating on SsMx-expressing cells were 100 times lower than those recorded on non-transfected cells. In contrast, SsMx did not inhibit ESV replication since no significant differences were observed regarding the virus yield or the major capsid protein gene transcription in transfected and non-transfected cells.

LGP2 expression is enhanced by interferon regulatory factor 3 in olive flounder, Paralichthys olivaceus.

In innate immunity, LGP2 (laboratory of genetics and physiology 2) plays a very important role in the production of type I interferon (IFN) through recognition of cytosolic viral RNA. Although viral infection or stimulation with double-strand RNA dramatically induces expression of the LGP2 gene, the underlying transcriptional mechanism has never been studied. Here, we cloned and characterized the 5'-upstream region (-1,337 bp) of the LGP2 gene in olive flounder (Paralichthys olivaceus). Numerous canonical motifs for IFN-regulatory factors (IRFs) were found in this region, and reporter assays identified a poly I:C-responsive promoter region (-506 to -398) that regulated LGP2 transcription. Transcriptional activity of the LGP2 promoter was strongly enhanced by IRF3, which bound to IRF3 motif #3 (-480). The LGP2 promoter was also responsive to viral infection in vitro. These results suggest that LGP2 transcriptional control is crucially involved to regulated by IRF3 function after viral infection or stimulation with poly I:C.

RNA aptamers inhibit the growth of the fish pathogen viral hemorrhagic septicemia virus (VHSV).

Viral hemorrhagic septicemia virus (VHSV) is a serious disease impacting wild and cultured fish worldwide. Hence, an effective therapeutic method against VHSV infection needs to be developed. Aptamer technology is a new and promising method for diagnostics and therapeutics. It revolves around the use of an aptamer molecule, an artificial ligand (nucleic acid or protein), which has the capacity to recognize target molecules with high affinity and specificity. Here, we aimed at selecting RNA aptamers that can specifically bind to and inhibit the growth of a strain of fish VHSV both in vitro and in vivo. Three VHSV-specific RNA aptamers (F1, F2, and C6) were selected from a pool of artificially and randomly produced oligonucleotides using systematic evolution of ligands by exponential enrichment. The three RNA aptamers showed obvious binding to VHSV in an electrophoretic mobility shift assay but not to other tested viruses. The RNA aptamers were tested for their ability to inhibit VHSV in vitro using hirame natural embryo (HINAE) cells. Cytopathic effect and plaque assays showed that all aptamers inhibited the growth of VHSV in HINAE cells. In vivo tests using RNA aptamers produced by Rhodovulum sulfidophilum showed that extracellular RNA aptamers inhibited VHSV infection in Japanese flounder. These results suggest that the RNA aptamers are a useful tool for protection against VHSV infection in Japanese flounder.

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.

Antiviral protection mechanisms mediated by ginbuna crucian carp interferon gamma isoforms 1 and 2 through two distinct interferon gamma-receptors.

Fish genomes possess three type II interferon (IFN) genes, ifnγ1, ifnγ2 and ifnγ-related (ifnγrel). The IFNγ-dependent STAT signalling pathway found in humans and mice had not been characterized in fish previously. To identify the antiviral functions and signalling pathways of the type II IFN system in fish, we purified the ifnγ1, ifnγ2 and ifnγrel proteins of ginbuna crucian carp expressed in bacteria and found them to elicit high antiviral activities against crucian carp hematopoietic necrosis virus. We also cloned two distinct ifnγ receptor alpha chain (ifngr1) isoforms, 1 and 2, and stably expressed them in HeLa cells by transfecting the cells with ifngr1-1 or ifngr1-2 cDNA. When receptor transfectants were treated with the ligands in a one-ligand-one-receptor manner (ifnγ1 and ifngr1-2 or ifnγ2 and ifngr1-1), the stat1 protein was phosphorylated at both serine-727 and tyrosine-701 residues. Gel shift mobility analysis and reporter assay clearly showed that the specific ligand-receptor interaction resulted in the binding of the stat1 protein to the GAS element and enhanced transcription. Therefore, the actions of ifnγ1 and ifnγ2 were found to be mediated by a specific receptor for each signalling pathway via a stat1-dependent mechanism.

In vitro evaluation of marine-microorganism extracts for anti-viral activity.

Viral-induced infectious diseases represent a major health threat and their control remains an unachieved goal, due in part to the limited availability of effective anti-viral drugs and measures. The use of natural products in drug manufacturing is an ancient and well-established practice. Marine organisms are known producers of pharmacological and anti-viral agents. In this study, a total of 20 extracts from marine microorganisms were evaluated for their antiviral activity. These extracts were tested against two mammalian viruses, herpes simplex virus (HSV-1) and vesicular stomatitis virus (VSV), using Vero cells as the cell culture system, and two marine virus counterparts, channel catfish virus (CCV) and snakehead rhabdovirus (SHRV), in their respective cell cultures (CCO and EPC). Evaluation of these extracts demonstrated that some possess antiviral potential. In sum, extracts 162M(4), 258M(1), 298M(4), 313(2), 331M(2), 367M(1) and 397(1) appear to be effective broad-spectrum antivirals with potential uses as prophylactic agents to prevent infection, as evident by their highly inhibitive effects against both virus types. Extract 313(2) shows the most potential in that it showed significantly high inhibition across all tested viruses. The samples tested in this study were crude extracts; therefore the development of antiviral application of the few potential extracts is dependent on future studies focused on the isolation of the active elements contained in these extracts.

The rainbow trout (Oncorhynchus mykiss) interferon response in the ovary.

Immune responses in the ovary are tightly regulated to provide protection for the developing germ cells, which are very sensitive to inflammatory responses. This characteristic immune response is often used by viral pathogens to evade the immune system, replicate and be transmitted to other specimens through the ovary. Taking into account that in teleost fish, the innate immune system is considered crucial to the outcome of viral infections and the interferon (IFN) system is considered as the first line of defence against viruses, we have studied the IFN response in rainbow trout (Oncorhynchus mykiss) ovary using two viruses with different replicative capacity in this organ, namely viral haemorrhagic septicaemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV). Both VHSV and IPNV are shed from the ovary, but while VHSV actively replicates at this site, IPNV remains silent. In this context, we have determined the levels of expression of IFNs and the IFN-induced Mx genes in the ovary upon in vivo and in vitro infections with VHSV and IPNV, and compared to the effects provoked by the viral mimic poly I:C in vivo. We have demonstrated that while VHSV strongly up-regulates all the IFN genes studied, IPNV in vivo exposure either has no effect or even provokes strong suppression of IFN gene expression. These differences are not observed in vitro, even though IPNV does not replicate actively in this case either. Finally, to better understand the role that the production of type I IFN plays in the ovary, we have studied the effects of two type I recombinant rainbow trout IFNs (rtIFN1 and rtIFN2) to modulate both the expression of immune genes and to establish an antiviral state in the ovary. Interestingly, the ovary was able to respond to both rtIFN1 and 2, despite the fact that the IFN1 gene was not expressed here. Moreover, rtIFN1 and rtIFN2 not only modulated the expression of genes related to the IFN response, but also modulated inflammatory genes and significantly suppressed VHSV replication.

Inhibitory effect of mycophenolic acid on the replication of infectious pancreatic necrosis virus and viral hemorrhagic septicemia virus.

Infectious pancreatic necrosis virus (IPNV) and viral hemorrhagic septicemia virus (VHSV) remain two of the most important pathogens of farmed trout worldwide. Mycophenolic acid (MPA) is an inhibitor of cellular inosine monophosphate dehydrogenase (IMPDH), an enzyme that catalyzes an essential step in the biosynthesis of GTP. In this report, the antiviral activity of MPA against IPNV and VHSV in cell culture was assessed. Cell viability, virus yield, protein and RNA synthesis determinations were used to evaluate the inhibitory effect of MPA. MPA caused a dose-dependent inhibition of IPNV and VHSV replication. It was found that MPA had a particularly potent effect against IPNV, inhibiting the production of infectious virus more than 10(5)-fold. MPA was also highly effective in preventing viral protein synthesis. Quantitative real-time RT-PCR was used to measure viral RNA in cells infected by IPNV or VHSV to evaluate the inhibitory capacity of MPA as well as to compare MPA to the established antiviral drug ribavirin. MPA showed a good efficacy in decreasing accumulation of viral RNA at low concentrations. Finally, time of addition and wash out experiments suggested that MPA may have a dual mechanism of action, targeting both a cell and a viral function. This study provides evidence that MPA can function as a broad-spectrum antiviral drug for use in therapy of rainbow trout diseases.

Teleostean IL11b exhibits complementing function to IL11a and expansive involvement in antibacterial and antiviral responses.

Interleukin 11 is a class-1 helical cytokine, having the four-helix bundle structure, possessing pleiotropic characteristics involved in physiological processes including blood production, bone formation and placentation. The interleukin 11 paralogues (IL11a and IL11b) have been identified in fish with only IL11a from carp and trout have been characterized and analyzed for its expression thus far. Here, we cloned and studied the structure and expression of IL11b in Japanese flounder (Paralichthys olivaceus), and compared this with the existing information on fish IL11 paralogues. Japanese flounder IL11b (poIL11b) cDNA is composed of 1536 bp encoding for 201 aa residues with a 23 aa leader peptide, three cysteine residues (C12, C183 and C198) and four potential N-linked glycosylation sites. poIL11b does not show constitutive expression in tissues of adult fish except for the very slight expression in kidney and spleen, and the very high expression in peripheral blood leukocytes (PBLs). poIL11b is transiently up-regulated by bacterial lipopolysaccharide (LPS) and increasingly stimulated by the IFN inducer poly I:C in kidney, spleen and peripheral blood leukocytes of adult fish in vitro. It is likewise slightly stimulated by Edwardsiella tarda infection but is highly expressed after hirame rhabdovirus (HIRRV) infection in kidney of juvenile fish. The stimulation studies suggest that poIL11b, aside from its role in bacterial infection, is well involved in antiviral responses. Moreover, poIL11b structure and expression pattern appears to be slightly distinct and opposite to IL11a, respectively, suggesting a complementation of function of the duplicate fish IL11 genes.

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.

Dual antiviral activity of human alpha-defensin-1 against viral haemorrhagic septicaemia rhabdovirus (VHSV): inactivation of virus particles and induction of a type I interferon-related response.

It is well known that human alpha-defensin-1, also designated as human neutrophil peptide 1 (HNP1), is a potent inhibitor towards several enveloped virus infecting mammals. In this report, we analyzed the mechanism of the antiviral action of this antimicrobial peptide (AMP) on viral haemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus. Against VHSV, synthetic HNP1 possesses two antiviral activities. The inactivation of VHSV particles probably through interfering with VHSV-G protein-dependent fusion and the inhibition of VHSV replication in target cells by up-regulating genes related to the type I interferon (IFN) response, such as Mx. Neither induction of IFN-stimulated genes (ISGs) by HNP1 nor their antiviral activity against fish rhabovirus has been previously reported. Therefore, we can conclude that besides to acting as direct effector, HNP1 acts across species and can elicit one of the strongest antiviral responses mediated by innate immune system. Since the application of vaccine-based immunization strategies is very limited, the used of chemicals is restricted because of their potential harmful impact on the environment and no antimicrobial peptides from fish that exhibit both antiviral and immunoenhancing capabilities have been described so far, HNP1 could be a model molecule for the development of antiviral agents for fish. In addition, these results further confirm that molecules that mediate the innate resistance of animals to virus may prove useful as templates for new antivirals in both human and animal health.