Functional nutrition modulates the early immune response against viral haemorrhagic septicaemia virus (VHSV) in rainbow trout.

Although viruses represent a major threat for cultured fish worldwide, the commercialization of vaccines capable of providing effective and long-lasting protection is still lacking for most of these viral diseases. In this situation, the use of supplemented diets could be a suitable strategy to increase the immune status of the fish and reduce the impact of viral pathogens. Among possible immunostimulants that could be included in these functional feeds, some studies have previously shown that certain ß-glucans can significantly increase certain immune parameters of fish and reduce the impact of viral diseases. However, the mechanisms through which ß-glucans exert their activity have not been fully elucidated yet. In the current study, we have studied the immune response of different tissues to viral haemorrhagic septicaemia virus (VHSV) in rainbow trout fed with a non-supplemented control diet as well as in fish fed a commercial functional aquafeed (Protec™, Skretting) containing ß-glucans, vitamin C, vitamin E and zinc. For this, after 30 days of feeding the fish with one of the two diets, they were subsequently infected with VHSV by bath or mock-infected. After 2 or 6 days post-infection, fish were sacrificed and the levels of transcription of different immune genes such as IgM, IgT, IgD, Mx, interferon γ (IFN γ) and perforin studied in different tissues (kidney, gut and gills). Additionally, the levels of natural IgMs in serum were also determined. Our results demonstrate that fish fed the functional diet were capable of mounting an increased IgM, IgT, IgD and Mx transcriptional response to the virus. Additionally, these fish also showed increased levels of natural IgMs in serum. These results reveal a previously undescribed effect of functional diets on fish Ig production and point to Protec™ as an adequate diet to be incorporated in holistic programs aimed at mitigating the effect of viral diseases.

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.

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.

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.

The olive leaf extract exhibits antiviral activity against viral haemorrhagic septicaemia rhabdovirus (VHSV).

A commercial plant extract derived from olive tree leaf (Olea europaea) (LExt) and its major compound, oleuropein (Ole), inhibited the in vitro infectivity of the viral haemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus. Incubation of virus with LExt or Ole before infection reduced the viral infectivity to 10 and 30%, respectively. Furthermore, LExt drastically decreased VHSV titers and viral protein accumulation (virucidal effect) in a dose dependent manner when added to cell monolayers 36 h post-infection. On the other hand, both the LExt and Ole were able to inhibit cell-to-cell membrane fusion induced by VHSV in uninfected cells, suggesting interactions with viral envelope. Therefore, we propose that O. europaea could be used as a potential source of promising natural antivirals, which have demonstrated to lack impact on health and environment. In addition, Ole could be used to design other related antiviral agents.