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.

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.

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.

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.

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.

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.