| Porcine reproductive and respiratory syndrome(Porcine Reproductive and Respiratory Syndrome,PRRSV),commonly known as blue ear pig,is an acute,contact,highly contagious disease caused by Porcine Reproductive and Respiratory Syndrome Virus(PRRSV).The main infections of the disease are pregnant sows and piglets.Pregnant sows show reproductive disorders,while piglets and their age-related pigs show severe respiratory symptoms and persistent infections.At present,the prevention and control of PRRSV in China mainly relies on vaccines.There are mainly two vaccines,attenuated vaccine and inactivated vaccine,but there is no specific antiviral drug against PRRSV.At present,the prevention and control of the disease in foreign countries is mainly to purify.The most classic method is to clear the group and build the group,but this will consume huge manpower and material resources.Therefore,there is an urgent need to find new ways to effectively prevent and control PRRSV and to develop drugs that are safe and effective against PRRSV.Traditional Chinese medicine has many kinds of resources,has the characteristics of small toxic and side effects and low drug resistance.Studies have shown that active ingredients with antiviral effects can be extracted from traditional Chinese medicines,some of which have significant inhibitory effects on PRRSV.Therefore,screening drugs with anti-PRRSV activity from traditional Chinese medicine has become a research hotspot.In this study,21 kinds of traditional Chinese medicines with antiviral activity,such as Stemona japonica(Blume)Miq.,Hedyotis diffusa and Lobelia chinensis Lour.,were selected.The traditional Chinese medicine extract were prepared by traditional water decoction method.The Marc-145 cell model was used to screen the extracts with anti-PRRSV effect in vitro,and the antiviral mechanism were further studied.Firstly,the maximum safe concentrations of Marc-145 cells(MNTC)were determined by thiazole blue colorimetric method(MTT),and ribavirin was used as a positive control drug.The maximum inactivations of PRRSV were screened by direct inactivation.The extracts of traditional Chinese medicine with high inhibition rate of PRRSV were screened out from the water extracts of traditional Chinese medicine with inhibition rate higher than 50%,and then the two action modes of blocking and inhibition were combined with cell morphology observation.The results showed that the extract of BJ(Because the subsequent research needs to publish and apply for a patent,the test uses the code to indicate the extract)has significant anti-PRRSV activity in vitro.The quality control of the extract of BJ was studied by thin layer chromatography and high performance liquid chromatography.The results of thin layer chromatography(TLC)identification of the extract of BJ showed that the chromatographic spots of the test and the control were clear,and the active ingredients in the extract of BJ were quickly identified.The results of high performance liquid chromatography(HPLC)showed that the established method has high sensitivity,strong specificity and good reproducibility,and is suitable for quality control of the extract of BJ.Used ribavirin as a positive control drug,the effects of the extract of BJ on PRRSV N gene synthesis and N protein expression at different time points were detected by fluorescence quantitative PCR and Western blot,respectively,and Annexin V-EGFP/PI double staining method was used to study the effect of the extract of BJ on apoptosis induced by PRRSV.In the experiment of the extract of BJ on the N gene,the results showed that the extract of BJ could inhibit the synthesis of N gene.Within 24 h～72 h,the N gene copy number of the extract of BJ group was significantly lower than that of the virus control group under the three modes of action(P<0.01),indicating that the extract of BJ could block the virus,inhibit virus replication,and has the ability to directly kill viruses.in the experiment,the effect of the extract of BJ on the content of N protein results showed that the extract of BJ could inhibit the expression of N protein.Within 24 h～72 h,the protein content of each group increased with time,but the N protein content of the extract was significantly lower than that of the virus control group(P<0.05).This result is consistent with the results of the gene level detection.Apoptosis test results showed that the late apoptosis rate of the extract of BJ in the three modes of action was not significantly different from that of the cell control group within 24 h～72 h,and the total apoptosis rate was significantly lower than that of the virus control group(P<0.05),indicating that the extract of BJ can inhibit apoptosis induced by PRRSV.This study found for the first time that the extract of BJ has an anti-PRRSV effect in vitro,and its antiviral mechanism is achieved by blocking virus adsorption,inhibiting viral replication,directly killing virus and inhibiting apoptosis induced by PRRSV.Moreover,the extract of BJ also exerts its anti-PRRSV mechanism by inhibiting N gene synthesis and N protein expression. |
