Effects And Mechanisms Of Deoxynivalenol On PRRSV Infection

Deoxynivalenol(DON)is a mycotoxin produced by Fusarium spp.DON is a natural compound with peculiar chemical structure,which makes it hard to synthetised.However,the commercial DON is so expensive that it can't meet the demands of large amount of toxicological researches.DON is toxic to animals or humans.Pigs are the most sensitive animals to DON as it markedly reduces feed intake and decreases weight gain.DON is also known to possess immunomodulatory properties,since high concentrations may result in immunosuppression,characterized by a decrease in circulating lymphocytes while low concentrations can induce stimulation of cytokine and inflammation associated genes.DON and other Fusarium mycotoxins have been shown to increase susceptibility to viral infections.Porcine Reproductive and Respiratory Syndrome Virus(PRRSV)is an important porcine infectious disease,causing severe reproductive impairment in breeding animals and respiratory problems in piglets.In this research,we prepared and purified DON mycotoxin under the existing conditions of our laboratory.The purified compound was used in the toxicological research.We have explored the effects of DON on the PRRSV replication in vitro.The effects of DON on the expression of cytokines and apoptosis in porcine alveolar macrophages(PAM)were studied.The in vivo study model of piglets infected with PRRSV was developed.Piglets in experimental group were feed with diet containing different concentrations of DON.Experiment 1 Separation and Purification of DON from Wheat CultureF.graminearum macroconidia of PH-1 strain was inoculated into wheat cultures.The yield of wheat culture was detected by HPLC.The ground wheat culture was extracted with acetonitrile/H2O(84:16,v/v).The brown sticky extract was developed by vibration,filtration,and evaporation.The extract was stirred with coarse silica gel and the first silica gel column chromatography.The petroleum ether,methylene chloride,ethyl acetate and methanol were used as the elution reagent.The target DON was in ethyl acetate detected by TLC.The liquid was concentrated to sticky extract and stirred with micromesh silica gel.The second silica gel column chromatography was developed with methylene chloride/methanol(19:1,v/v)as the elution reagent.The extract of DON samples was performed by preparative HPLC.The single peak was collected and analyzed on HPLC.The retention time was identical to that of the DON standard.The chemical structure of the compound was identified by HPLC-MS and NMR.The immunochemical analysis of the compound was developed on a competitive ELISA.The compound was proved to be DON.In this study,433 mg of DON extract was developed from 1 kg wheat culture and 293 mg of purified DON was obtained.The recovery rate was approach to 70%.The purity of the DON was higher than 90%which met the requirement of toxicological researches.Experiment 2 Effects of DON on PRRSV replication in vitroIn this study,the MARC-145 cells were chosen to cultivate PRRSV to study the effects of DON on PRRSV replication in vitro.Cells infected with PRRSV or non-infected with PRRSV were treated with different concentrations of DON.The cell viability and LDH activity of both infected and non-infected cells were detected.The antigen of PRRSV and the virus titer in DON treated cells were detected.The effects of DON on PRRSV N protein replication were studied.It turned out that the high concentration of DON(500?800 ng·mL-1)displayed a lower cell viability(P<0.05)and a higher LDH activity(P<0.05)in non-infected cells.The cellular morphology was also changed by the high concentration of DON.In this study,the concentrations of DON lower than 500 ng·mL-1 were studied on the effects of PRRSV replication.For PRRSV infected cells,cytopathic effects(CPE)were caused by the virus.The CPE of cells treated with 50 ng·mL-1 DON was similar to the cells with no DON.PRRSV infected cells treated with 150?200 ng·mL-1 DON displayed a higher viability(P<0.05)and a lower LDH activity(P<0.05)than control group.The PRRSV antigen and virus titer was cut down at the DON concentration of 150?200 ng·mL-1.The N protein replication was inhibited by 150?200 ng·mL-1 DON,showed in the Western blotting experiment.The rusults suggested that PRRSV replicaion in vitro was effected by DON at the concentration of 150?200 ng·mL-1.Experiment 3 Effects of DON on the cytokines expression and apoptosis of cells infected with PRRSVPAM is the natural target cell of PRRSV infection.For further study of the effects of DON on PRRSV replication,the cytokine mRNA expression and apoptosis of PAM were studied.The cytokines included antiviral cytokines(IFN-?and IFN-?)and pro-inflammatory cytokines(IL-1?and IL-6).Results showed that IFN-?and IFN-?of cells treated with 200 ng·mL-1 DON had no significant difference with the control group.PRRSV infected cells had a higher expression of IFN-a and IFN-?than control group,but the difference was not significant.The relative expression of IFN-?and IFN-?in PRRSV infected cells treated with DON were significantly higher than that of the control group(P<0.05).It is suggested that the increase expression of antiviral cytokines was related to the inhibition of DON on PRRSV.Results showed that DON could significantly(P<0.05)increase the relative expression of IL-1?and IL-6.PRRSV could increase the relative expression of IL-1?and IL-6 with no significant difference compared to the control group.DON could increase the relative expression of IL-1?and IL-6 in PRRSV infected cells,which was significantly higher than that of other groups(P<0.05).The increase expression of pro-inflammatory cytokines induced by DON may played a role in the inhibition of PRRSV replication.In this study,the apoptosis of DON on PRRSV infected cells was detected by flow cytometry.For PRRSV infected cells,the apoptosis rate of DON at the concentration of 50 ng·mL-1 was similar to that of the control group.The apoptosis rate of cells treated with 150?200 ng·mL-1 DON was significantly higher than that of control group(P<0.05).It is suggested that the apoptosis caused by DON at concentrations of 150?200 ng·mL-1 maybe the reason that threatened the survival of PRRSV in cells.Experiment 4 In vivo study of DON on PRRSV infected pigletsIn order to study the effects of DON on PRRSV infected piglets,28 days of weaned piglets were divided into 4 groups randomly.Each group owned 10 piglets,which were segregated feed.Group I was control group,piglets were feed with basal diet.Piglets in other groups were intramuscularly inoculated and intranasally inoculated with 1 mL of JXA1 strain cell culture fluids(TCID50 1×105·mL-1),respectively.Piglets in Group 2 were feed with basal diet.Piglets in Group 3 and Group 4 were feed with basal diet containing 3 mg·kg-1 and 5 mg·kg-1 of DON.The growth performance,body temperature,virus load in blood and lungs and the antibody response of serum were tested.The antigen content in lung samples was assayed by Immunohistochemistry.Results showed that piglets fed with feed containing DON had low growth performance,high body temperature,high virus load in blood and lungs.The antigen content in lung samples of DON treated group was higher than that of control group,assayed by Immunohistochemistry.The antibody response against PRRSV in serum samples was enhanced by DON addition,according to the serum ELISA test.Anorectic effects of DON were additive to those of PRRSV.The results of this study were not in consistent with that of the in vitro study,which may be explained by the different environment of in vitro and in vivo study.Another reason was that the level of DON addition in vivo study was lower than that of the PRRSV replication inhibition.The main effects of DON in vivo were food refusal and viomit.