Preparation Of Red Elemental Selenium (Se~0) And Its Antiviral Activity Against Spring Viremia Of Carp Virus (SVCV)

Selenium(Se),as a nonmetallic element of the chalcogens,is an essential trace element for plants,mammals and human health.As its large surface area,selenium has strong adsorption and anti-oxidation,such as anti-hydroxyl radicals,anti-DNA oxidation and anti-microbial infection.It has been found that selenium has potential applications in immune regulation,antioxidation,anti-aging,inhibiting tumors and anti-microbial infections.The hydroxyl radicals scavenging activity of elemental nano-selenium is 5-fold of inorganic selenium and 2.5-fold of organic selenium,and its toxicity is only 1/7 of selenite.In antiviral activity,the studying has only been involved in a few viruses related to human diseases,such as anti-human herpes virus,influenza virus,encephalomyocarditis virus,vesicular stomatitis virus.In this study,we used Herbaspirillum sp.WT00C to produce red elemental selenium(Se~0)via fermentation,and the red elemental selenium purified from the fermentation culture was used to culture EPC cells or feed crucian carp and zebrafish.The antiviral activity of red elemental selenium against SVCV was systematically studied at the levels of cell and living fishes,and the main conclusions are as follows:1.Efficient large-scale production of red elemental selenium and optimization of production conditions.After Herbaspirillum sp.WT00C was incubated in LB medium containing 200 m M sodium selenate at 37℃for 28 hours,the bacterium was harvested and renamed as Herbaspirillum sp.WT00C-se.Herbaspirillum sp.WT00C-se was inoculated into the LB medium containing 200 m M sodium selenate and fermented at 37℃.The optimality conditions obtained from comprehensive tests ware:the selenate concentration for bacterial activation was 75 m M and for fermentation was 200 m M.The fermentation was performed at37℃,200 rpm,p H 7.0 and maximum voluntary ventilation of 1 v/v.min.The yield of red elemental selenium was 560 mg/L.These results provide a strongly technical support for producing red elemental selenium by using Herbaspirillum sp.WT00C in the follow-up fermentation production.2.Red elemental selenium effectively improves the antiviral capability of EPC cells.After EPC cells were co-cultured with different concentrations of red elemental selenium,MTT assay showed that 5-20μg/ml red elemental selenium did not display any toxicity towards EPC cells.q-PCR showed that 5-20μg/ml red elemental selenium effectively stimulated the expression of IFN and ISG15 genes.TCID50 assay showed that the virus titers were significant lower in EPC cells growing in 5,10,20μg/ml red elemental selenium for 48 hours after SVCV infection.Different concentrations of red elemental selenium were mixed with SVCV virus for 12 hours,and then infected EPC cells.The virus titers did not change compared to the same virus without treated with the red elemental selenium.This result indicated that the red elemental selenium did not directly interact with the virus particles.In other words,the red elemental selenium did not directly affect viral Infectivity.These results suggest that the antiviral effect of red elemental selenium comes from cell metabolism of selemiun,rather than red elemental selenium directly interacts with SVCV virus.3.Red elemental selenium improves the antiviral capability of crucian carp and promotes the growth of crucian carp.The red elemental selenium was uniformly mixed with the fish food to prepare a selenium-containing food of 0-10μg/g,and the fires of crucian carp were fed with the fish food containing 0-10μg/g selenium for 60 days.After SVCV infection,the morbidities and mortalities were observed and counted.Meanwhile,the expression of IFN and SVCV-N genes were detected.The results showed that only 3 fishes were died in the control group and no fish was died in other experimental groups.The expression of SVCV-N gene in the control group was much higher than that in the other experimental groups.Meanwhile,the expression of IFN gene in the groups of 5-10μg/g selenium was 13-and 39-fold increase respectively compared to the control group.In addition,the average carcass growth of crucian carp fed with fish food containing 0,0.5,5,10μg/g selenium was 1.92,1.96,2.25,2.73 cm,and the average weight was 1.78,1.77,2.18,3.38 g after feeding 4 months.In the group fed with fish food containing 10μg/g selenium,the length and weight of fish body showed a statistically significant increase.4.Red elemental selenium improves antiviral activity of zebrafish.The fish food red worm was uniformly mixed with different concentrations of red elemental selenium to prepare a selenium-containing food of 0-10μg/g selenium,and the fires of zebrafish were fed with the red worm containing 0-10μg/g selenium for 60 days.After SVCV infection,the morbidities and mortalities was observed and counted,and the expression of IFN gene was detected.Compared to the control group,the survival rate of the zebrafish fed with the 5μg/g selenium was significantly increased.The expression of IFN gene in the groups of 5 and 10μg/g selenium was also significantly increased.However,the survival rate in the group of 10μg/g selenium was significantly lower than that of the control group.The q-PCR analysis showed that the expression of IRFs,Mx,TNF-α,IL-1β,and IL-12 genes were significantly increased in the group of 10μg/g selenium.The overexpression of these inflammatory factors suggests that strong immune and inflammatory responses occur in the fish group of 10μg/g selenium under the dual effects of SVCV infection and 10μg/g selenium,which finally results in more fish death.In this project,the large-scale production of red elemental selenium is achieved via fermentation of Herbaspirillum sp.WT00C-se and the production conditions are also optimized.The red elemental selenium purified from bacterial fermentation culture was co-cultured with EPC cells or fed with crucian carp and zebrafish,and then the antiviral activity was examined after SVCV infection.All results obtained from either EPC cells or crucian carp and zebrafish demonstrate that the properly selenium contributes to improving the antiviral activity of fish,whereas excess selenium causes excessive immune and inflammatory responses.These experimental results not only provide a theoretical basis and technical support for the commercial and green production of red elemental selenium but also for applying red elemental selenium to improve the anti-SVCV virus capability of fish.