Rhein: A potent immunomodulator empowering largemouth bass against MSRV infection.

Micropterus salmoides rhabdovirus (MSRV) is a significant viral pathogen in largemouth bass aquaculture, causing substantial annual economic losses. However, effective prevention methods remain elusive for various reasons. Medicinal plant extracts have emerged as valuable tools in preventing and managing aquatic animal diseases. Thus, the search for immunomodulators with straightforward, safe structures in plant extracts is imperative to ensure the continued health and growth of the largemouth bass industry. In our research, we employed epithelioma papulosum cyprinid (EPC) cells and largemouth bass as models to assess the anti-MSRV properties and immunomodulatory effects of ten plant-derived bioactive compounds. Among them, rhein demonstrated noteworthy potential, exhibiting a 75 % reduction in viral replication in vitro at a concentration of 50 mg/L. Furthermore, rhein pre-treatment significantly inhibited MSRV genome replication in EPC cells, with the highest inhibition rate reaching 64.8 % after 24 h, underscoring rhein's preventive impact against MSRV. Likewise, rhein displayed remarkable therapeutic effects on EPC cells during the early stages of MSRV infection, achieving a maximum inhibition rate of 85.6 % in viral replication. Subsequent investigations unveiled that rhein, with its consistent activity, effectively mitigated cytopathic effects (CPE) and nuclear damage induced by MSRV infection. Moreover, it restrained mitochondrial membrane depolarization and reduced the apoptosis rate by 38.8 %. In vivo experiments reinforced these findings, demonstrating that intraperitoneal injection of rhein enhanced the expression levels of immune related genes in multiple organs, hindered virus replication, and curtailed the mortality rate of MSRV-infected largemouth bass by 29 %. Collectively, our study endorses the utility of rhein as an immunomodulator to combat MSRV infections in largemouth bass. This not only underscores the potential of rhein as a broad-spectrum antiviral and means to bolster the immune response but also highlights the role of apoptosis as an immunological marker, making it an invaluable addition to the armamentarium against aquatic viral pathogens.

Application of rhein as an immunostimulant controls spring viremia of carp virus infection.

In recent years, the exploration of natural compounds possessing both immunostimulatory and antiviral activities has attracted growing attention in aquaculture research. Consequently, the pursuit of identifying natural products exhibiting anti-SVCV potential as immunostimulants holds significant promise, offering a pathway to mitigate the economic ramifications inflicted by SVCV outbreaks in aquaculture settings. Among them, rhein emerges as a particularly compelling contender. Boasting a widespread distribution, well-established extraction methods, and multiple biological activities, it has exhibited the capacity to enhance the antiviral activity of host cells in vitro by blocking the viral internalization process, with a peak inhibition rate of 44.0%. Based on this intervention, rhein inhibited apoptosis and mitochondrial damage triggered by SVCV infection, ultimately producing a significant antiviral effect. Moving beyond the laboratory setting, rhein's efficacy translates effectively into in vivo scenarios. It has demonstrated substantial antiviral potency by increasing the expression of antiviral-related genes, most notably, retinoic acid-inducible gene I (RIG-I), interferon-φ (IFN-φ) and IFN-stimulated gene product 15 (ISG15). In concert with this genetic modulation, rhein efficiently reduces the viral load, precipitating a consequential enhancement in the survival rate of SVCV-infected fish, elevating it to an encouraging 16%. In conclusion, the outcomes of our investigation offer a compelling testament to rhein's potential as a valuable immunomodulator in the battle against SVCV infections in aquaculture, and the remarkable attributes exhibited by rhein underscore its viability for future commercial deployment.

Long-term exposure to azoxystrobin induces immunodeficiency in fish that are vulnerable to subsequent rhabdovirus infection.

Azoxystrobin (AZ) is one of the most widely used strobilurin fungicides in the world, and its residue has seriously endangered aquatic ecological security. Our previous data showed that AZ exposure may reduce the resistance of fish to rhabdovirus infection in aquatic environment. Here, we further reported a potential long-term adverse effect of AZ exposure on the antiviral and immunosuppressive recovery in fish, and observed that mitochondrial dynamic balance was disturbed by AZ in which excessive mitochondrial fission occurred in response to decreased ATP levels. When a recovery operation was performed in AZ-exposed cells and fish, infectivity of our model virus, spring viraemia of carp virus (SVCV), was significantly decreased in vitro (using the epithelioma papulosum cyprini [EPC] fish cell line) and in vivo (using zebrafish) in a time-dependent manner. Also, the expression of eight innate antiviral immune genes (IFNs, ISG15, MX1, RIG-I, IRF3, Nrf2 and HO-1) showed a similar change to SVCV replication between the longer exposure period and the expression recovery. Additionally, AZ facilitated horizontal transmission of SVCV in a static cohabitation challenge model, predicting the increase of the potential for the viral outbreak. Therefore, our data suggest that long-term effect of AZ on irreparable impairment in fish made AZ residue potentially greater for ecological risks.

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.

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.

Azoxystrobin increases the infection of spring viraemia of carp virus in fish.

Azoxystrobin (AZ) has entered aquatic ecosystems and produced serious damages to fish associated with potentially increasing the susceptibility to pathogens. This study characterized the defense abilities of fish by exposed to AZ on challenging with the infection of spring viraemia of carp virus (SVCV). The results showed that SVCV replication increased significantly in EPC cells and zebrafish that were exposed to up to 50 µg/L of AZ at 3, 5, 7, and 14 d. Intracellular biochemical assays indicated that AZ at 5 and 50 µg/L inhibited the activation of Nrf2-ARE pathway including a decrease in Nrf2 expression, Nrf2 phosphorylation, HO-1 content, and three antioxidant activities. While no significant difference in ERK1/2 and JNK MAPKs in zebrafish was observed, P38 phosphorylation was significantly decreased at 7 and 14 d, and the changes in MAPKs were more evident in EPC cells previously exposed to AZ at 7 d. These results revealed that AZ initially induced low phosphorylation of MAPKs, triggering the attenuation of Nrf2 phosphorylation to weaken Nrf2 translocation into the nucleus in a longer exposure period (more than 5 d). The data in the cells and fish also showed that antioxidant activities were decreased to some extent at 5-7 d for the cells and 7-14 d for the fish. Furthermore, interferon-related factors were decreased in AZ-exposed zebrafish, explaining the reason that fish can't resist the virus infection. Overall, the present study provided a new adverse threat of AZ by amplifying the viral outbreak to endanger ecological safety in aquatic environment.

Potential aquatic environmental risks of trifloxystrobin: Enhancement of virus susceptibility in zebrafish through initiation of autophagy.

Chronic pollution in aquatic ecosystems can lead to many adverse effects, including a greater susceptibility to pathogens among resident biota. Trifloxystrobin (TFS) is a strobilurin fungicide widely used in Asia to control soybean rust. However, it has the potential to enter aquatic ecosystems, where it may impair fish resistance to viral infections. To explore the potential environmental risks of TFS, we characterized the antiviral capacities of fish chronically exposed to TFS and subsequently infected with spring viraemia of carp virus (SVCV). Although TFS exhibited no significant cytotoxicity at the tested environmental concentrations during viral challenge, SVCV replication increased significantly in a time-dependent manner within epithelioma papulosum cyprini (EPC) cells and zebrafish exposed to 25 µg/L TFS. Results showed that the highest viral load was more than 100-fold that of the controls. Intracellular biochemical assays indicated that autophagy was induced by TFS, and associated changes included an increase in autophagosomes, conversion of LC3-II, accumulation of Beclin-1, and degradation of P62 in EPC cells and zebrafish. In addition, TFS markedly decreased the expression and phosphorylation of mTOR, indicating that activation of TFS may be associated with the mTOR-mediated autophagy pathway. This study provides new insights into the mechanism of the immunosuppressive effects of TFS on non-target aquatic hosts and suggests that the existence of TFS in aquatic environments may contribute to outbreaks of viral diseases.

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.

Inhibition of a novel coumarin on an aquatic rhabdovirus by targeting the early stage of viral infection demonstrates potential application in aquaculture.

Spring viremia of carp virus (SVCV) is one of the most serious pathogens in aquaculture, resulting in devastating damage in cyprinid. In this study, we designed and synthesized a novel coumarin derivative (C3007) for evaluating its in vitro and in vivo anti-SVCV effects. Here, we determined that up to 25 mg/L C3007 significantly decreased SVCV protein gene expression levels in EPC cells by a maximum inhibitory rate of >95%. When C3007 was preincubated with SVCV, infectivity was significantly inhibited in vitro in a time-dependent manner, with complete inhibition at 25 mg/L. For in vivo studies, C3007 exhibited an anti-SVCV effect by substantially enhancing the survival rate of virus-infected fish via intraperitoneal injection. Although the horizontal transmission of SVCV was hindered by C3007 in a static cohabitation challenge model, it was not completely blocked, showing that the viral loads in recipient fish were obviously reduced. Thus, C3007 could potentially be used as a therapeutic agent with great potential in aquatic systems and may also be suitable for applications in pond aquaculture settings against viral transmission. Additionally, the C3007-preincubated virus induced an antiviral immune response with high levels of IFN expression, suggesting that C3007 pre-treatment could be used in vaccine development.

PMA-triggered PKCε activity enhances Nrf2-mediated antiviral response on fish rhabdovirus infection.

Viral infection is often accompanied with alteration of intracellular redox state, especially an imbalance between reactive oxygen species (ROS) production and antioxidant cellular defenses. The previous studies showed that an antioxidant cellular defense system, the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), played an important role against spring viraemia of carp virus (SVCV) infection in fish. To further reveal the mediated mechanism that Nrf2 active state was affected by protein kinase C (PKC), here we evaluated SVCV replication in host cells by treated with a strong activator of PKC phorbol-12-myristate-13-acetate (PMA) and an inhibitor staurosporine. Our results showed that PMA significantly repressed SVCV replication and viral-induced apoptosis in Epithelioma papulosum cyprini (EPC) cell, suggesting that PKC may exhibit an anti-SVCV effect. Likewise, PMA resulted in a higher phosphorylation levels of PKCε rather than PKCα/ß to participate in the activation of Nrf2, mainly involved in the activation of Nrf2 phosphorylation of Ser40 to favor Nrf2 translocation to nucleus. Furthermore, the data revealed that PMA up-regulated an antiviral response heme oxygenase-1 (HO1) gene expression that was confirmed as the key player against SVCV infection by HO1 specific siRNA. Overall, this study provided a new therapeutic target for the treatment of SVCV infection, and modulating PKC activity could be used for the prevention and treatment of SVCV.