Herbal active small molecule as an immunomodulator for potential application on resistance of common carp against SVCV infection.

Herbal immunomodulators are an important part of prevention and control on viral diseases in aquaculture because of their propensity to improve immunity in fish. The present study was conducted to evaluate the immunomodulatory effect and antiviral activity of a synthesized derivative (serial number: LML1022) against spring viremia of carp virus (SVCV) infection in vitro and in vivo. The antiviral data suggested that LML1022 at 100 µM significantly inhibited the virus replication in epithelioma papulosum cyprini (EPC) cells, and may completely inhibit the infectivity of SVCV virion particles to fish cells by affecting the viral internalization. The results in the related stability of water environments also demonstrated that LML1022 had an inhibitory half-life of 2.3 d at 15 °C, which would facilitate rapid degradation of LML1022 in aquaculture application. For in vivo study, the survival rate of SVCV-infected common carp was increased 30% at least under continuous oral injection of LML1022 at 2.0 mg/kg for 7 d treatment. Additionally, pretreatment of LML1022 on fish prior to SVCV infection also obviously reduced the viral loads in vivo as well as an improved survival rate, showing that LML1022 was potential as an immunomodulator. As an immune response, LML1022 significantly upregulated the immune-related gene expression including IFN-γ2b, IFN-I, ISG15 and Mx1, indicating that its dietary administration may improve the resistance of common carp against SVCV infection.

Antiviral effects of natural small molecules on aquatic rhabdovirus by interfering with early viral replication.

Spring viremia of carp virus (SVCV) is globally widespread and poses a serious threat to aquatic ecology and aquaculture due to its broad host range. To develop effective agents to control SVCV infection, we selected 16 naturally active small molecules to assess their anti-SVCV activity. Notably, dihydroartemisinin (DHA) (100 µmol/L) and (S, S)-(+)-tetrandrine (TET) (16 µmol/L) exhibited high antiviral effects in epithelioma papulosum cyprinid (EPC) cells, with inhibitory rates of 70.11% and 73.54%, respectively. The possible antiviral mechanisms were determined as follows: 1. Pre-incubation with DHA and TET decreased viral particle infectivity in fish cells, suggesting that horizontal transmission of SVCV in the aquatic environment was disrupted; 2. Although neither had an effect on viral adhesion, TET (but not DHA) interfered with SVCV entry into host cells (>80%), suggesting that TET may have an antiviral function in early viral replication. For in vivo study, both agents enhanced the survival rate of SVCV-infected zebrafish by 53.3%, significantly decreased viral load, and modulated the expression of antiviral-related genes, indicating that DHA and TET may stimulate the host innate immune response to prevent viral infection. Overall, our findings indicated that DHA and TET had positive effects on suppressing SVCV infection by affecting early-stage viral replication, thus holding great potential as immunostimulants to reduce the risk of aquatic rhabdovirus disease outbreaks.

In vitro and in vivo inhibition of a novel arctigenin derivative on aquatic rhabdovirus.

Spring viraemia of carp virus (SVCV) poses a serious threat to aquaculture industry due to the lack of approved antiviral treatments. Therefore, a novel arctigenin derivative, 4-(2-methylimidazole) octanoxy-arctigenin (MON), was synthesized to assess the antiviral activity against SVCV in vitro and in vivo. The results indicated MON decreased the SVCV glycoprotein (G) gene expression in vitro by a maximum inhibitory rate of > 99% at 3.5 µM. Furthermore, MON showed the protective effect on epithelioma papulosum cyprinid (EPC) cells and considerably decreased the cytopathic effect (CPE). More importantly, MON inhibited SVCV G gene expression levels in vitro at the half-maximal activity (IC50) of 0.18 µM at 48 h. For in vivo studies, MON demonstrated anti-SVCV activity by enhancing the survival rate of zebrafish (Danio rerio) after infection via pelvic fin base injection. These results tended to be consistent with MON decreasing the SVCV titer of infected zebrafish. During this time, viral loads of the spleen and kidney have declined in SVSV-infected zebrafish. Based on the histopathological assay, MON exhibited the high protective effect in the spleen and kidney of SVCV-infected fish. Combined, MON is on track to become a novel agent to address SVCV infection in aquaculture.

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.

Synthesis and biological evaluation of novel coumarin derivatives in rhabdoviral clearance.

Diseases caused by rhabdoviruses have had a huge impact on the productive lives of the entire human population. The main problem is the lack of drugs for the treatment of this family of viruses. Infectious hematopoietic necrosis virus (IHNV), the causative agent of IHN, is a typical rhabdovirus which has caused huge losses to the salmonid industry. Therefore, in this study, IHNV was studied as a model to evaluate the antiviral activity of 35 novel coumarin derivatives. Coumarin A9 was specifically selected for further validation studies upon comparing the half maximum inhibitory concentration (IC50) of four screened candidate derivatives in epithelioma papulosum cyprinid (EPC) cells, as it exhibited an IC50 value of 2.96 µM against IHNV. The data revealed that A9 treatment significantly suppressed the virus-induced cytopathic effect (CPE) in EPC cells. In addition, A9 showed IC50 values of 1.68 and 2.12 µM for two other rhabdoviruses, spring viremia of carp virus and micropterus salmoides rhabdovirus, respectively. Furthermore, our results suggest that A9 exerts antiviral activity, but not by destroying the virus particles and interfering with the adsorption of IHNV. Moreover, we found that A9 had an inhibitory effect on IHNV-induced apoptosis in EPC cells, as reflected by the protection against cell swelling, formation of apoptotic bodies, and loss of cell morphology and nuclear division. There was a 19.05 % reduction in the number of apoptotic cells in the A9 treatment group compared with that in the IHNV group. In addition, enzyme activity assays proved that A9 suppressed the expression of caspase 3, 8 and 9. These results suggested that A9 inhibit viral replication, to some extent, by blocking IHNV-induced apoptosis. In an in vivo study, A9 exhibited an anti-rhabdovirus effect in virus-infected fish by substantially enhancing the survival rate. Consistent with the above results, A9 repressed IHNV gene expression in virus-sensitive tissues (brain, kidney and spleen) in the early stages of virus infection. Importantly, the data showed that horizontal transmission of IHNV was reduced by A9 in a static cohabitation challenge model, especially in fish that underwent bath treatment, suggesting that A9 might be a suitable therapeutic agent for IHNV in aquaculture. Therefore, coumarin derivatives can be developed as antiviral agents against rhabdoviruses.

Red elemental selenium (Se0 ) improves the immunoactivities of EPC cells, crucian carp and zebrafish against spring viraemia of carp virus.

Selenium, as an essential trace element, interferes through selenoproteins in many physiological processes of plants and mammals. Its antiviral activity has recently attracted much attention because selenium improves the antiviral capacity of animal cells against a few viruses relevant to human diseases. In this study, the red elemental selenium was purified from the fermentative culture of Herbaspirillum camelliae WT00C and then used to culture epithelioma papulosum cyprinid (EPC) cells or feed crucian carp and zebrafish. Finally, its antiviral effects were investigated at the cell level and living fishes after spring viraemia of carp virus infection. At the cell level, 5, 10 and 20 µg ml-1 red elemental selenium significantly induced the expression of interferon (IFN) and ISG15 genes in EPC cells. The viral TCID50 (50% tissue culture infective dose) values in the EPC cells incubated with 5, 10 and 20 µg ml-1 red elemental selenium were significantly less than those of the control. More expression of IFN and ISG15 genes and less TCID50 values indicate that red elemental selenium indeed improves the antiviral capability of EPC cells. In the crucian carp fed with the food containing 5 and 10 µg g-1 red elemental selenium, IFN expressions showed 13- and 39-fold increases at the 16th day of post-injection, and its expression was dependent on selenium concentrations. Meanwhile, no fish death occurred in all the experimental groups. In the zebrafish fed with the red worm containing 5 µg g-1 red elemental selenium, IFN and Mx expressions and survival rate were significantly higher than those of the control. The results of this study show that red elemental selenium indeed improves the antiviral activity of fish. The antiviral effects of selenium mainly come from its immune regulation through its incorporation into selenoproteins. The optimum level of selenium contributes to improving fish immunity, whereas excess selenium causes excessive immune and inflammatory responses.

In vitro and in vivo evaluation of antiviral activity of a phenylpropanoid derivative against spring viraemia of carp virus.

Phenylpropanoids, common natural compounds, possess many different biological activities such as antioxidant, anti-inflammatory and antiviral. Spring viraemia of carp virus (SVCV) can cause a high mortality in common carp (Cyprinus carpio). However, there are currently no licenced drugs that effectively cure this disease. In this study, we designed and synthesized a phenylpropanoid derivative 4-(4-methoxyphenyl)-3,4-dihydro-2H-chromeno[4,3-d]pyrimidine-2,5(1 H)-dione (E2), and explored the antiviral effect against SVCV in vitro and in vivo. Up to 25 mg/L of E2 significantly inhibited the expression levels of SVCV protein genes in the epithelioma papulosum cyprini (EPC) cell line by a maximum inhibitory rate of >90%. As expected, E2 remarkably declined the apoptotic of SVCV-infected cells and suppressed potential enhancement of the mitochondrial membrane potential (ΔΨm), these data implied that E2 could protect mitochondria from structural damage in response to SVCV. Meanwhile, E2 was added to EPC cells under four different conditions: time-of-addition, time-of-removal, pre-treatment of viruses and pre-treatment of cells indicated that E2 may block the post-entry transport process of the virus. Additionally, the up-regulation of six interferon (IFN)-related genes also demonstrated that E2 indirectly activated IFNs for the clearance of SVCV in common carp. Drug cure effect showed that treatment with E2 at 0.5 d post infection (dpi) is more effective than at 0, 1 or 2 dpi. Most importantly, intraperitoneal therapy of E2 markedly improved common carp survival rate and reduced virus copies in body. Therefore, the E2 has potential to be developed into a novel anti-SVCV agent.

Hydroxycoumarin efficiently inhibits spring viraemia of carp virus infection in vitro and in vivo.

Spring viremia of carp virus (SVCV) causes devastating losses in aquaculture. Coumarin has an advantageous structure for the design of novel antiviral agents with high affinity and specificity. In this study, we evaluated a hydroxycoumarin medicine, i.e., 7-(6-benzimidazole) coumarin (C10), regarding its anti-SVCV effects in vitro and in vivo. Results showed that up to 12.5 mg/L C10 significantly inhibited SVCV replication in the epithelioma papulosum cyprini (EPC) cell line, with a maximum inhibitory rate of >97%. Furthermore, C10 significantly reduced cell death and relieved cellular morphological damage in SVCV-infected cells. Decreased mitochondrial membrane potential (ΔΨm) also suggested that C10 not only protected mitochondria, but also reduced apoptosis in SVCV-infected cells. For in vivo studies, intraperitoneal injection of C10 resulted in an anti-SVCV effect and substantially enhanced the survival rate of virus-infected zebrafish. Furthermore, C10 significantly enhanced antioxidant enzyme activities and decreased reactive oxygen species (ROS) to maintain antioxidant-oxidant balance within the host, thereby contributing to inhibition of SVCV replication. The up-regulation of six interferon (IFN)-related genes also demonstrated that C10 indirectly activated IFNs for the clearance of SVCV in zebrafish. This was beneficial for the continuous maintenance of antiviral effects because of the low viral loads in fish. Thus, C10 is suggested as a therapeutic agent with great potential against SVCV infection in aquaculture.

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.

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.

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.

Inhibition of an Aquatic Rhabdovirus Demonstrates Promise of a Broad-Spectrum Antiviral for Use in Aquaculture.

Many enveloped viruses cause devastating disease in aquaculture, resulting in significant economic impact. LJ001 is a broad-spectrum antiviral compound that inhibits enveloped virus infections by specifically targeting phospholipids in the lipid bilayer via the production of singlet oxygen (1O2). This stabilizes positive curvature and decreases membrane fluidity, which inhibits virus-cell membrane fusion during viral entry. Based on data from previous mammalian studies and the requirement of light for the activation of LJ001, we hypothesized that LJ001 may be useful as a preventative and/or therapeutic agent for infections by enveloped viruses in aquaculture. Here, we report that LJ001 was more stable with a prolonged inhibitory half-life at relevant aquaculture temperatures (15°C), than in mammalian studies at 37°C. When LJ001 was preincubated with our model virus, infectious hematopoietic necrosis virus (IHNV), infectivity was significantly inhibited in vitro (using the epithelioma papulosum cyprini [EPC] fish cell line) and in vivo (using rainbow trout fry) in a dose-dependent and time-dependent manner. While horizontal transmission of IHNV in a static cohabitation challenge model was reduced by LJ001, transmission was not completely blocked at established antiviral doses. Therefore, LJ001 may be best suited as a therapeutic for aquaculture settings that include viral infections with lower virus-shedding rates than IHNV or where higher viral titers are required to initiate infection of naive fish. Importantly, our data also suggest that LJ001-inactivated IHNV elicited an innate immune response in the rainbow trout host, making LJ001 potentially useful for future vaccination approaches. IMPORTANCE: Viral diseases in aquaculture are challenging because there are few preventative measures and/or treatments. Broad-spectrum antivirals are highly sought after and studied because they target common components of viruses. In our studies, we used LJ001, a broad-spectrum antiviral compound that specifically inhibits enveloped viruses. We used the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV) as a model to study aquatic enveloped virus diseases and their inhibition. We demonstrated inhibition of IHNV by LJ001 both in cell culture as well as in live fish. Additionally, we showed that LJ001 inhibited the transmission of IHNV from infected fish to healthy fish, which lays the groundwork for using LJ001 as a possible therapeutic for aquatic viruses. Our results also suggest that virus inactivated by LJ001 induces an immune response, showing potential for future preventative (e.g., vaccine) applications.

VSV replication in neurons is inhibited by type I IFN at multiple stages of infection.

Vesicular stomatitis virus (VSV) is a rhabdovirus which causes acute encephalitis in mice after intranasal infection. Because type I interferon (IFN) has been shown to be a potent inhibitor of VSV, we investigated the role of type I IFN in viral replication in neurons in culture. Pre-treatment of NB41A3 neuroblastoma cells or primary neuron cultures with IFN-beta or IFN-alpha strongly inhibits virus replication, with 1000-fold inhibition of infectious virus release occurring at 7 h post-infection, and maximum inhibition of 14,000-fold occurring at 14 h. Type I IFN inhibited both viral protein and RNA synthesis, but not enough to account for the inhibition of infectious virus yield. The influenza virus protein NS1 binds dsRNA and antagonizes induction of PKR activity, an IFN-inducible antiviral protein which phosphorylates and inactivates the elongation factor eIF-2alpha, resulting in cessation of translation. In NS1-expressing neuroblastoma cells, VSV replication was inhibited by IFN-beta as well as in control NB41A3 cells, and eIF-2alpha phosphorylation was blocked, suggesting that PKR activity was not involved in inhibition of viral protein synthesis. Similarly, inhibition of VSV by IFN-beta was not affected by addition of inhibitors of nitric oxide synthase, indicating that IFN-beta activity is not mediated by nitric oxide or superoxide. This contrasts with the essential role of NOS-1 in inhibition of VSV replication when neurons are treated with IFN-gamma. Analysis of cell culture supernatants revealed suppression of release of VSV particles from both NB41A3 cells and primary neurons treated with IFN. The inhibition of virion release closely matched the overall suppression of infectious VSV particle release, suggesting that type I IFN plays a role in inhibition of VSV assembly.

The role of interleukin-18 in vesicular stomatitis virus infection of the CNS.

Intranasal application of vesicular stomatitis virus (VSV) results in the initial infection of the olfactory receptor neurons and a rapid progression of the virus through the mouse central nervous system (CNS). Interleukin-18 (IL-18) is an 18.3-kd cytokine that induces interferon gamma (IFN-gamma) production in mice. IL-18 is synthesized as an inactive precursor that is cleaved and activated by caspase-1/interleukin-1beta converting enzyme (ICE). IL-18 shares several biological properties with IL-12, including the ability to induce IFN-gamma production in T lymphocytes and natural killer (NK) cells. In the CNS, microglia and astrocytes produce IL-18 and IL-12. We have previously shown that IL-12 promotes recovery from VSV encephalitis. This led us to examine the potential role of IL-18 in the pathogenesis of VSV encephalitis. We show that both IL-18 and caspase-1 mRNA are consistently present in the CNS of mice. The addition of exogenous IL-18 to cell cultures does not affect the production of VSV, and addition of exogenous IL-18 at the time of infection does not alter the morbidity or mortality of BALB/c mice. In vitro studies with neutralizing monoclonal antibody to IL-18 had no effect. From these results we conclude that in this system and under the experimental conditions used, unlike IL-12 and IFN-gamma, IL-18 does not play a significant role in the host response to VSV infection.

Interleukin-12 promotes recovery from viral encephalitis.

Infusion of interleukin-12 (IL-12) enhances recovery from lethal experimental vesicular stomatitis virus (VSV) infection of the central nervous system (CNS). Interleukin-12 treatment resulted in: 1) increased survival frequency; 2) faster recovery from weight loss; 3) substantially decreased VSV titers in brain homogenates and diminished immunohistochemical detection of VSV antigens in tissue sections; 4) earlier and increased CNS expression of types 1, 2, and 3 nitric oxide synthase (NOS) and both major histocompatibility complex (MHC) class I and class II antigens; 5) earlier and increased blood and CNS levels of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). These results suggest that IL-12 enhances recovery from VSV infection of the CNS.