Evaluation of the effects of Astragalus polysaccharides as immunostimulants on the immune response of crucian carp and against SVCV in vitro and in vivo.

This experiment was conducted to evaluate the immunomodulatory effect and antiviral activity of Astragalus polysaccharides (APS) in crucian carp and epithelioma papulosum cyprinid (EPC) cells. Two diets containing 0 and 2 g/kg, APS were fed crucian carp for 56 days. The results showed that supplementation with APS significantly upregulated the immune-related indices including the levels of IgM, the activities of LZM, AKP and ACP, and the contents of C3 and C4. At the same time, compared with the CK group, adding APS to the feed significantly upregulated the expression of IL-8, IL-10, IL-1ß, IFN-α, IFN-γ, MyD88, TGF-ß and TNF-α in the spleen, kidney, liver and intestine of crucian carp. In addition, when the crucian carp were injected with SVCV, the survival rates of fish in the APS group and the control group were 48.87% and 13.76%, respectively. These results indicated that dietary APS could improve the resistance of crucian carp against SVCV infection. APS also significantly decreased viral titer and inhibited apoptosis induced by SVCV in EPC cells. These results indicated that APS could stimulate the immune response of crucian carp and improve the abilities of crucian carp and EPC cells to resist SVCV infection.

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.

Suppression effect of plant-derived berberine on cyprinid herpesvirus 2 proliferation and its pharmacokinetics in Crucian carp (Carassius auratus gibelio).

Cyprinid herpesvirus 2 (CyHV-2), which infects silver crucian carp including goldfish (Carassius auratus auratus) and Crucian carp (Carassius auratus gibelio) with high mortality, is an emerging viral pathogen worldwide. Previous studies showed that berberine (BBR), a bioactive plant-derived alkaloid, demonstrated potential antiviral actions against many different viruses. Here, we assessed the effect of berberine hydrochloride (BBH) on the replication of CyHV-2 in vitro and in vivo. Cytotoxicity assay indicated that 5-25 µg/mL BBH was non-toxic to the RyuF-2 cells. In viral inhibition assays, real time PCR was employed to titrate the genomic copy number of progeny virus, real time RT-PCR was applied to monitor the transcriptional levels of viral genes, and Western blot analysis was performed to detect the synthetic levels of viral proteins. The results demonstrated that BBH systematically impedes the viral gene transcription and suppressed the replication of CyHV-2 in RyuF-2 cells. In animal challenge test, BBH was confirmed to protect Crucian carps from CyHV-2 infection in a dose-dependent manner, which was supported by suppressed viral replication levels, reduced viral pathogenesis and higher survival rates. Furthermore, pharmacokinetics data of BBH in Crucian carp revealed its rapid absorption (Tmax of 1.5 h), suitable plasma half-life (t1/2z/h of 7-12 h depending on oral dosage), and dose-dependent drug exposure properties following oral administration (revealed by AUC0-t values). These findings shed light on repurposing BBH to treat CyHV-2 infections in silver crucian carp.

Highly efficient inhibition of spring viraemia of carp virus replication in vitro mediated by bavachin, a major constituent of psoralea corlifonia Lynn.

As one of nine piscine viruses recognized by the International Office of Epizootics, spring viraemia of carp virus (SVCV) is an important pathogen bringing high mortality to cyprinids. Up to now, there is no approved therapy on SVCV, making them strong public health threat in aquaculture. In this study, the anti-SVCV activities of 12 plant crude extracts were investigated by using epithelioma papulosum cyprini (EPC) cells. Among these plants, Psoralea corylifolia Linn. showed the highest inhibition on SVCV replication, with an inhibitory percentage of 67.98%. Further studies demonstrated that bavachin (BVN), one of the major constituents of Psoralea corylifolia Linn., was also highly effective to SVCV infection. The half maximal inhibitory concentrations (IC50) of BVN on SVCV glycoprotein and nucleoprotein expression were 0.46 (0.29-0.73) and 0.31 (0.13-0.55) mg/L, respectively. In addition, SVCV-induced apoptosis which may be negative to SVCV replication was inhibited by BVN. The apoptotic cells were decreased 21.42% for BVN compared with SVCV group. These results indicated that the inhibition of BVN on SVCV replication was, in some extent, via blocking SVCV induced apoptosis. Furthermore, cellular morphological damage induced by SVCV was also blocked by BVN treatment. Mechanistically, BVN did not affect SVCV infectivity and cannot be used for prevention of SVCV infection. Time-of-addition and viral binding assays revealed that BVN mainly inhibited the early events of SVCV replication but did not interfere with SVCV adsorption. In conclusion, BVN was considered to develop as a promising agent to treat SVCV infection.

A specific CpG oligodeoxynucleotide induces protective antiviral responses against grass carp reovirus in grass carp Ctenopharyngodon idella.

CpG oligodeoxynucleotides (ODNs) show strong immune stimulatory activity in vertebrate, however, they possess specific sequence feature among species. In this study, we screened out an optimal CpG ODN sequence for grass carp (Ctenopharyngodon idella), 1670A 5'-TCGAACGTTTTAACGTTTTAACGTT-3', from six published sequences and three sequences designed by authors based on grass carp head kidney mononuclear cells and CIK (C. idella kidney) cells proliferation. VP4 mRNA expression was strongly inhibited by CpG ODN 1670A in CIK cells with GCRV infection, showing its strong antiviral activity. The mechanism via toll-like receptor 9 (TLR9)-mediated signaling pathway was measured by real-time quantitative RT-PCR, and TLR21 did not play a role in the immune response to CpG ODN. The late up-regulation of CiRIG-I mRNA expression indicated that RIG-I-like receptors (RLRs) signaling pathway participated in the immune response to CpG ODN which is the first report on the interaction between CpG and RLRs. We also found that the efficient CpG ODN can activates interferon system. Infected with GCRV, type I interferon expression was reduced and type II interferon was induced by the efficient CpG ODN in CIK cells, especially IFNγ2, suggesting that IFNγ2 played an important role in response to the efficient CpG ODN. These results provide a theoretical basis and new development trend for further research on CpG and the application of CpG vaccine adjuvant in grass carp disease control.

Characterizations of two grass carp Ctenopharyngodon idella HMGB2 genes and potential roles in innate immunity.

High-mobility group box 2 (HMGB2) protein is a chromatin-associated nonhistone protein, involved in transcriptional regulation and nucleic-acid-mediated innate immune responses in mammalian. However, the function of piscine HMGB2 in innate immune responses is still unknown. In the present study, two HMGB2 homologue genes (CiHMGB2a, CiHMGB2b) were identified and characterized in grass carp (Ctenopharyngodon idella). Both CiHMGB2a and CiHMGB2b genes encode proteins with 213 amino acids, sharing 71.4% identities and containing two basic HMG boxes and an acidic tail. The deduced protein sequences showed the most identities to HMGB2a (93%) and HMGB2b (86.4%) of zebrafish (Danio rerio), respectively. Quantitative real-time RT-PCR (qRT-PCR) analysis showed that CiHMGB2a and CiHMGB2b were constitutively expressed in all the 15 tested tissues. Post grass carp reovirus (GCRV) infection, mRNA levels of CiHMGB2a and CiHMGB2b were strongly up-regulated in spleen and head kidney and mildly modulated in C. idella kidney (CIK) cells. Meanwhile, mRNA expressions of CiHMGB2a and CiHMGB2b were significantly regulated by viral pathogen associated molecular patterns (PAMPs) polyinosinic-polycytidylic potassium salt (poly(I:C)) and bacterial PAMPs lipopolysaccharide (LPS), peptidoglycan (PGN) challenge in CIK cells. In CiHMGB2a and CiHMGB2b over-expression cells, expressions of CiHMGB2a and CiHMGB2b facilitated each other; transcription levels of CiTRIF, CiMyD88, CiIPS-1 and CiMx1 were remarkably enhanced, whereas CiIFN-I was inhibited, compared with those in cells transfected with pCMV (control plasmid); after GCRV challenge, all those tested genes were up-regulated with divergent expression profiles. Antiviral activities of CiHMGB2a and CiHMGB2b were manifested by the delayed appearance of cytopathic effect (CPE) and inhibition of GCRV yield. All those results demonstrate that CiHMGB2a and CiHMGB2b not only mediate antiviral immune responses but also involve in responding to viral/bacterial PAMPs challenge, which provides novel insights into the essential role of HMGB2 in innate immunity.

Molecular cloning and immune responsive expression of a ribonuclease III orthologue involved in RNA interference, dicer, in grass carp Ctenopharyngodon idella.

In this study, the dicer gene (designated as cidicer) was identified and characterized from grass carp Ctenopharyngodon idella. The complementary DNA (cDNA) of cidicer contained an open reading frame (ORF) of 5646 nucleotides (nts) encoding a putative protein of 1881 amino acids (aa). The deduced Dicer protein contained all known functional domains identified in other organisms. Tissue tropism analysis indicated that cidicer is abundantly expressed in brain, gill, head kidney, liver, spleen, heart, muscle and intestine. In the C. idella kidney (CIK) cells, messenger RNA (mRNA) expression of cidicer was significantly up-regulated at 24 h (6·36-fold, P < 0·01) after grass carp reovirus (GCRV) infection, and its transcriptional expression level was also transiently induced to a high level (6·54-fold, P < 0·01) at 2 h post-stimulation of synthetic double-stranded polyinosinic-polycytidylic potassium salt [poly(I:C)]. In vivo analysis further showed that the expression of cidicer mRNA in the liver was induced to a significantly high level at 12 h (8·46-fold, P < 0·01), and then dropped to normal level at 72 h post-challenge with GCRV. The transcriptional expression pattern of cidicer in the spleen tissue was similar to that of liver tissue upon GCRV challenge. These results collectively implied that the identified cidicer was an inducible gene responding to viral infection both in vitro and in vivo, and the data would shed light on the interaction between RNA interference (RNAi) antiviral pathway and aquareovirus infection.