Vitamin D3 can effectively and rapidly clear largemouth bass ranavirus by immunoregulation.

Largemouth bass ranavirus (LMBV) is a highly destructive pathogen that causes significant mortality rates among largemouth bass populations. Unfortunately, there is a dearth of drug development efforts specifically aimed at treating LMBV. To address this, our study sought to investigate the potential effectiveness of incorporating varying doses of VD3 into the diet as a treatment for LMBV. Through qRT-PCR and semi-qPCR, we observed significant suppression and clearance of LMBV pathogens in largemouth bass fed with 15000 IU/Kg and 20000 IU/Kg of VD3 within 14 days. In addition, VD3 treatment significantly increased the expression levels of key immune-related genes such as IL-1ß, IFN-γ, Mx, and IgM. Encouragingly, we observed that VD3 significantly increased antioxidant and immune activities such as TSOD, TAOC and C3 in serum and maintained total protein levels. Additionally, tissue pathology sections highlighted a dose-dependent relationship between VD3 supplementation and tissue damage, with the 15000 IU and 20000 IU groups exhibiting minimal damage. In conclusion, a reasonable concentration of VD3 effectively reduced LMBV replication and tissue damages, while improved immune-related genes expression and serum biochemical indices. These findings declare the considerable therapeutic potential of VD3 supplementation for combating LMBV disease and provide an alternative treatment option for fish farming.

Antiviral Activities of Green Tea Components against Grouper Iridovirus Infection In Vitro and In Vivo.

(1) Background: Singapore grouper iridovirus (SGIV) can cause extensive fish deaths. Therefore, developing treatments to combat virulent SGIV is of great economic importance to address this challenge to the grouper aquaculture industry. Green tea is an important medicinal and edible plant throughout the world. In this study, we evaluated the use of green tea components against SGIV infection. (2) Methods: The safe working concentrations of green tea components were identified by cell viability detection and light microscopy. Additionally, the antiviral activity of each green tea component against SGIV infection was determined with light microscopy, an aptamer (Q5c)-based fluorescent molecular probe, and reverse transcription quantitative PCR. (3) Results: The safe working concentrations of green tea components were green tea aqueous extract (GTAE) ≤ 100 µg/mL, green tea polyphenols (TP) ≤ 10 µg/mL, epigallocatechin-3-gallate (EGCG) ≤ 12 µg/mL, (-)-epigallocatechin (EGC) ≤ 10 µg/mL, (-)-epicatechin gallate (EGC) ≤ 5 µg/mL, and (-)-epicatechin (EC) ≤ 50 µg/mL. The relative antiviral activities of the green tea components determined in terms of MCP gene expression were TP > EGCG > GTAE > ECG > EGC > EC, with inhibition rates of 99.34%, 98.31%, 98.23%, 88.62%, 73.80%, and 44.31%, respectively. The antiviral effect of aptamer-Q5c was consistent with the results of qPCR. Also, TP had an excellent antiviral effect in vitro, wherein the mortality of fish in only the SGIV-injection group and TP + SGIV-injection group were 100% and 11.67%, respectively. (4) Conclusions: In conclusion, our results suggest that green tea components have effective antiviral properties against SGIV and may be candidate agents for the effective treatment and control of SGIV infections in grouper aquaculture.

Research on the indirect antiviral function of medicinal plant ingredient quercetin against grouper iridovirus infection.

Grouper iridovirus is a devastating pathogen that belongs to the genus Ranavirus. Based on the previous results that natural ingredient quercetin isolated from Illicium verum Hook. f. could effectively inhibit Singapore grouper iridovirus (SGIV) replication, suggesting that quercetin could serve as potential antiviral agent against grouper iridovirus. To know about whether quercetin has indirect antiviral activity against SGIV, this study made the investigation in vitro and in vivo, and the potential mechanism was also explored. Pretreating the cells with quercetin (12.5 µg/mL) significantly inhibited the replication of SGIV, similar results were also confirmed in vivo. Importantly, quercetin pretreatment could induce the expression of genes involved in type I interferon (IFN) system (IFN, STAT1, PKR, MxI and ISG15) and TLR9. It suggested that quercetin exerted the indirect antiviral activity against SGIV infection through promoting the recognition of SGIV and activating the IFN pathway to establish the antiviral status of host cell. Taken together, our results shedded light on the indirect antiviral function of natural ingredient quercetin, and clearly demonstrated that natural ingredient quercetin will be an excellent potential agent against SGIV infection in grouper aquaculture.

Antiviral activity of Illicium verum Hook. f. extracts against grouper iridovirus infection.

Grouper iridovirus causes high mortality rates in cultured groupers, and effective treatment for grouper iridovirus infection is urgently required. Illicium verum Hook. f. is a well-known medicinal plant with a variety of biological activities. The aim of this study was to analyse the use of I. verum extracts to treat grouper iridovirus infection. The safe working concentration of each I. verum extract was identified both in vitro and in vivo as follows: I. verum aqueous extract (IVAE) ≤ 500 µg/ml; I. verum ethanol extract (IVEE) ≤ 250 µg/ml; shikimic acid (SKA) ≤ 250 µg/ml; trans-anethole (TAT) ≤ 800 µg/ml; 3,4-dihydroxybenzoic acid (DDBA) ≤ 400 µg/ml; and quercetin (QCE) ≤ 50 µg/ml. The inhibitory activity of each I. verum extract against grouper iridovirus infection was analysed using aptamer (Q2)-based fluorescent molecular probe (Q2-AFMP) and RT-qPCR. All of the I. verum extracts displayed dose-dependent antiviral activities against grouper iridovirus. Based on the achieved per cent inhibition, IVAE, IVEE, DDBA and QCE were associated with the greatest antiviral activity (all > 90%). Together, our results indicate that I. verum extracts have effective antiviral properties, making it an excellent potential source material for the development of effective treatment for grouper iridovirus infection.

Effects of hesperidin on the growth performance, antioxidant capacity, immune responses and disease resistance of red swamp crayfish (Procambarus clarkii).

We evaluated the effects of hesperidin on the nonspecific immunity, antioxidant capacity and growth performance of red swamp crayfish (Procambarus clarkii). A total of 900 healthy crayfish were randomly divided into six groups: the control group (fed the basal diet) and the HES25, HES50, HES75, HES100 and HES150 groups, which were fed the basal diet supplemented with 25, 50, 75, 100 and 150 mg kg-1 hesperidin, respectively. The feeding experiment lasted 8 weeks. The results indicated that compared with the control group, the crayfish groups supplemented with 50-150 mg kg-1 hesperidin had a decreased feed conversion ratio (FCR) and increased final body weight (FBW), specific growth rate (SGR) and weight gain (WG) (P < 0.05). The protein carbonyl content (PCC), reactive oxygen species (ROS) level and malondialdehyde (MDA) level in the hepatopancreas and hemocytes were significantly lower, while the total antioxidant capacity (T-AOC), glutathione peroxidase (GPx) activity, and superoxide dismutase (SOD) activity were significantly higher in the crayfish groups supplemented with 50-150 mg kg-1 hesperidin than in the control group. Supplementation with 50-150 mg kg-1 hesperidin significantly increased the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), lysozyme (LZM), and phenoloxidase (PO) compared with the control group (P < 0.05); upregulated the mRNA expression of cyclophilin A (CypA), extracellular copper-zinc superoxide dismutase (ecCuZnSOD), GPxs, crustin, astacidin, Toll3 and heat shock protein 70 (HSP70) (P < 0.05); and decreased crayfish mortality following white spot syndrome virus (WSSV) infection. These findings indicate that dietary hesperidin supplementation at an optimum dose of 50-150 mg kg-1 may effectively improve nonspecific immunity, antioxidant capacity and growth performance in crayfish.

OsHV-1 infection leads to mollusc tissue lesion and iron redistribution, revealing a strategy of iron limitation against pathogen.

The mass mortality of molluscs caused by OsHV-1 infection has frequently occurred worldwide in recent years. Meanwhile the interaction between OsHV-1 and its host is largely unknown. Innate immunity mainly makes up the mollusc defense system, due to the lack of adaptive immunity in invertebrates. The iron limitation strategy is an indispensable facet of innate immunity across vertebrate and invertebrate species. In this study, an iron limitation strategy was interestingly found to contribute to mollusc innate immune responses against OsHV-1 infection. Firstly, ark clams, Scapharca broughtonii, were experimentally infected with OsHV-1, and serious hyperaemia in hepatopancreases and the erosion of gills were observed post OsHV-1 infection according to a histology assay. Meanwhile, based on quantification and Prussian blue staining, the process of iron efflux from ark clams was described post OsHV-1 infection. Secondly, ferritin, as an important iron storage protein, was characterized in ark clams and showed significant iron binding activity. According to the results of an immunohistochemistry assay, ferritin was supposed to be responsible for the iron translocation in ark clams post OsHV-1 infection. Its expression level was significantly fluctuant in response to OsHV-1 infection. Finally, oxidative stress was assessed by the analyses of H2O2 content, total antioxidant capacity and MDA level post OsHV-1 infection. Supplementary iron was found to promote ROS generation and death of hemocytes in vivo. These results highlighted that microenvironment changes in the essential nutrient iron should be an important aspect of the pathogenesis of OsHV-1 disease.

Antiviral function of grouper MDA5 against iridovirus and nodavirus.

Melanoma differentiation-associated gene 5 (MDA5) is a critical member of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family which can recognize viral RNA and enhances antiviral response in host cells. In this study, a MDA5 homolog from orange spotted grouper (Epinephelus coioides) (EcMDA5) was cloned, and its roles on grouper virus infection were characterized. The full-length EcMDA5 cDNA encoded a polypeptide of 982 amino acids with 74% identity with MDA5 homolog from rock bream (Oplegnathus fasciatus). Amino acid alignment analysis indicated that EcMDA5 contained three functional domains: two caspase activation and recruitment domain (CARDs), a DEAD box helicase-like (DExDc) domain, a helicase superfamily C-terminal domain (HELICc), and a C-terminal regulatory domain (RD). Upon challenge with Singapore grouper iridovirus (SGIV) or polyinosin-polycytidylic acid (poly I:C), the transcript of EcMDA5 was significantly up-regulated especially at the early stage post-injection. Under fluorescence microscopy, we observed that EcMDA5 mostly localized in the cytoplasm of grouper spleen (GS) cells. Interestingly, during virus infection, the distribution pattern of EcMDA5 was significantly altered in SGIV infected cells, but not in red spotted grouper nervous necrosis virus (RGNNV) infected cells, suggested that EcMDA5 might interact with viral proteins during SGIV infection. The ectopic expression of EcMDA5 in vitro obviously delayed virus infection induced cytopathic effect (CPE) progression and significantly inhibited viral gene transcription of RGNNV and SGIV. Moreover, overexpression of EcMDA5 not only significantly increased interferon (IFN) and IFN-stimulated response element (ISRE) promoter activities in a dose dependent manner, but also enhanced the expression of IRF3, IRF7 and TRAF6. In addition, the transcription level of the proinflammatory factors, including TNF-α, IL-6 and IL-8 were differently altered by EcMDA5 overexpression during SGIV or RGNNV infection, suggesting that the regulation on proinflammatory cytokines by EcMDA5 were also important for RGNNV infection. Together, our results demonstrated for the first time that the inhibitory effect of fish MDA5 on iridovirus replication might be mainly through the regulation of proinflammatory cytokines.

Detection of Torque teno sus virus types 1 and 2 by nested polymerase chain reaction in sera of sows at parturition and of their newborn piglets immediately after birth without suckling colostrum and at 24 hr after suckling colostrum.

This study was performed to clarify the sow-to-fetus transmission pathway of Torque teno sus virus (TTSuV) types 1 (TTSuV1) and 2 (TTSuV2). For this purpose, detection of TTSuV1 and TTSuV2 (TTSuVs) in sera of 6 sows (Sows 1-6) at parturition and in sera of their newborn piglets immediately after birth without suckling colostrum was performed by nested polymerase chain reaction (nPCR). These sows were bred using semen that had tested negative for TTSuVs. In a TTSuV1- and TTSuV2-positive sow (Sow 1), TTSuV1 and TTSuV2 were detected in 4 and 5 of 12 newborn littermates, respectively. In a TTSuV1-positive sow (Sow 2), TTSuV1 was detected in 1 of 8 newborn littermates. In 4 TTSuV1- and TTSuV2-negative sows (Sows 3-6), TTSuV1 was detected in 6 out of the 25 newborn piglets of 3 sows (Sows 3-5), while TTSuVs were not detected in all 13 piglets of 1 sow (Sow 6). In addition, to investigate the possibility of a sow-to-piglet transmission pathway of TTSuV via colostrum, TTSuV1 and TTSuV2 in sera of 12 newborn piglets from Sows 1-3 were examined by nPCR. Immediately after birth without suckling colostrum, TTSuV1 and TTSuV2 were not detected in 10 and 8 of 12 newborn piglets, respectively; however, at 24 hr after suckling colostrum, TTSuV1 was detected in 6 piglets, while TTSuV2 was not detected in any piglets. These results confirmed the existence of a sow-to-fetus transmission pathway of TTSuV during normal pregnancy and suggested a possibility of sow-to-piglet transmission of TTSuV via colostrum.

Effect of probiotics enriched diet on Paralichthys olivaceus infected with lymphocystis disease virus (LCDV).

We report the effect of two probiotics, Lactobacil and Sporolac as separate or mixed diets on innate immune mechanisms, such as phagocytosis activity, superoxide anion production of blood leukocytes, complement activity and plasma lysozyme activity, and disease resistance in lymphocystis disease virus (LCDV) infected olive flounder, Paralichthys olivaceus (523.5 +/- 18.3 g) on week 1, 2, and 4. In infected fish, administration of diet supplemented with Lactobacil individually or mixed with Sporolac significantly enhanced the immune parameters like phagocytic activity superoxide anion production, complement activity, and plasma lysozyme. However administration of supplemented diet with Sporolac alone, all the chosen immune parameters did not enhance when compared to control group; this diets resulted in lower mortality (30% and 25%) than Sporolac diet group (45%) in 30 days. We conclude that Lactobacil individually or mixed with Sporolac act as immunostimulants that enhance the innate immune response and disease resistance in lymphocystis disease virus (LCDV) infected olive flounder.

Effect of Punica granatum solvent extracts on immune system and disease resistance in Paralichthys olivaceus against lymphocystis disease virus (LDV).

We report the effect of aqueous, ethanol, and methanol solvent leaf extracts of Punica granatum on innate immune mechanisms, such as phagocytosis activity, respiratory burst activity, alternative complement activity, lysozyme activity and functional immunity in terms of percentage cumulative mortality and Relative Percent Survival (RPS) in olive flounder Paralichthys olivaceus naturally infected with lymphocystis disease virus (LDV) after 8 weeks. Infected fish were intraperitoneally administered with 0, 5, 50, and 100 mg kg(-1) body weight of solvent extracts. In groups treated with 50 and 100 mg kg(-1) body weight, the chosen innate immune parameters significantly increased after 8 weeks when compared to 0 mg kg(-1) dose, but not with 5 mg kg(-1). Administration of P. granatum solvent extracts for 8 weeks significantly reduced the percentage mortality with the consequent increase in RPS. The results suggest that intraperitoneal administration of the leaf extracts of P. granatum at 50 or 100 mg kg(-1) dose clearly enhance the innate immune responses and disease resistance after 8 weeks in P. olivaceus against natural LDV infection.

Evidence of Torque teno virus (TTV) vertical transmission in swine.

Routes of swine torque teno virus (TTV) transmission have been minimally investigated in the pig population. Current knowledge suggests the faecal-oral route as the most probable way of viral dissemination. Other transmission routes, such as mother-to-infant, have been studied in humans, but no information is available for swine. Thus, the objective of the present study was to determine the prevalence of two swine TTV genogroups, TTV1 and TTV2, in colostrum samples (n=61) and sera samples from sows (n=10) and stillborn pigs coming from them (n=30). Colostrum was fractioned into two components, milk whey and cell pellets, and 26 out of 61 milk whey samples and 30 out of 58 cell pellets analyzed contained TTV1 or TTV2 genomes, respectively, detected by specific PCR methods. Six and 3 out of 10 serum samples from sows were positive for TTV1 and TTV2 DNA, respectively. Finally, 15 out of 30 sera from stillborns were PCR positive for TTV1, but only 2 were TTV2 positive. Positive stillborns were always infected with the same TTV genogroup as their mothers. However, TTV sequence analysis determined that sequences obtained from sows and their stillborns were not identical. In conclusion, our results indicated that swine TTVs can be transmitted vertically, and suggest that different sow-to-piglet transmission routes may coexist, including transplacental/intra-uterine as well as through lactation. This study represents the first description of swine TTV presence in colostrum and stillborn piglets.

Studies on the immunomodulatory effect of extract of Cyanodon dactylon in shrimp, Penaeus monodon, and its efficacy to protect the shrimp from white spot syndrome virus (WSSV).

The present study investigates the protection of shrimp Penaeus monodon against white spot syndrome virus (WSSV) using antiviral plant extract derived from Cyanodon dactylon and the modulation of the shrimp non-specific immunity. To determine the antiviral activity, the shrimp were treated by both in vitro (intramuscular injection) and in vivo (orally with feed) methods at the concentration of 2mg per animal and 2% of the plant extract incorporated with commercially available artificial pellet feed, respectively. The antiviral activity of C. dactylon plant extract was confirmed by PCR, bioassay and Western blot analysis. In the present study, anti-WSSV activity of C. dactylon plant extract by in vivo and in vitro methods showed strong antiviral activity and the immunological parameters such as proPO, O(2)(-), NO, THC and clotting time were all significantly (P<0.05) higher in the WSSV-infected shrimp treated with plant extract when compared to control groups. These results strongly indicate that in vivo and in vitro administration of C. dactylon plant extract enhances immunity of the shrimp. Based on the present data and the advantages of plant extract available at low price, we believe that oral administration of C. dactylon plant extract along with the pellet feed is a potential prophylactic agent against WSSV infection of shrimp.

Dietary sodium alginate administration affects fingerling growth and resistance to Streptococcus sp. and iridovirus, and juvenile non-specific immune responses of the orange-spotted grouper, Epinephelus coioides.

The percent weight gain (PWG) and feeding efficiency (FE) of fingerling orange-spotted grouper, Epinephelus coioides, fed diets containing sodium alginate at 1.0 and 2.0 g kg(-1) were calculated on the 2nd, 4th, 6th, and 8th weeks after feeding. Survival rates of the fingerling grouper against Streptococcus sp. and an iridovirus, and non-specific immune parameters such as alternative complement activity (ACH50), lysozyme activity, natural haemagglutination activity, respiratory bursts, superoxide dismutase (SOD) activity, and phagocytic activity of juvenile grouper were also determined when the fish were fed diets containing sodium alginate at 0.5, 1.0, or 2.0 g kg(-1). The PWG and FE of fish were better when the fish were fed diets containing sodium alginate at 1.0, and 1.0 and 2.0 g kg(-1), respectively. The PWG and FE of fish fed the 0, 1.0 and 2.0 g kg(-1) sodium alginate-containing diets after 8 weeks were 271.0%, 454.4% and 327.8%, and 0.61, 0.72 and 0.68, respectively. Fish fed a diet containing sodium alginate at the level of 2.0 g kg(-1) had a significantly higher survival rate than those fed the control diet after challenge with Streptococcus sp. and an iridovirus causing an increase of survival rate by 25.0% and 16.7%, respectively, compared to the control group. The ACH(50) level of fish fed the sodium alginate-containing diets at 2.0 g kg(-1) was significantly higher than those fed the 1.0 g kg(-1) sodium alginate diet and control diet after 12 days, and had increased to 1.9-fold, compared to those fed the control diet. The lysozyme activity, phagocytic activity, respiratory bursts, and SOD level of fish fed the sodium alginate-containing diets at 1.0 and 2.0 g kg(-1) were significantly higher than those fed the control diet after 12 days, and had increased to 1.97- and 1.68-fold, 1.35- and 1.50-fold, 1.63- and 1.81-fold, and 1.23- and 1.31-fold, respectively, compared to those fed the control diet. We therefore recommend dietary sodium alginate administration at 1.0 and 2.0 g kg(-1), respectively, to promote growth and enhance immunity and resistance against Streptococcus sp. and an iridovirus.