Baicalin And Carazoles Inhibit Pseudorabies Virus Replication

Pseudorabies virus（PRV）continues to circulate in China’s pig herd and is a pathogen that is very harmful to the pig industry.In 2011,PRV variants appeared in China,resulting in a large-scale outbreak of PRV in China,which brought huge economic losses to China’s pig industry.Flavonoids and carbazoles have been shown to have broad-spectrum antiviral activity.Among them,the antiviral effects of a large number of flavonoids such as baicalin,quercetin,hesperidin and catechin have been studied in depth.In this study,the biological activity of baicalin and carbazole compounds against PRV was evaluated at the cellular level,which laid the foundation for the development of PRV antiviral drugs.The specific research content is as follows:（1）In this study,polyclonal antibodies to PRV US3 and EP0 were first prepared and used for subsequent experiments.In this study,the toxicity of baicalin to PRV and the efficiency of inhibiting PRV replication were evaluated at the cellular level:PK-15 cells were treated with different concentrations of baicalin,and the 50%cytotoxicity concentration(CC₅₀)measured with the CCk-8kit was 152.9μM;In the process of PRV infection,different concentrations of baicalin were added,and the toxicity of PRV was determined 24 h after infection,and the results showed that baicalin could significantly inhibit the in vitro replication of PRV and was dose-dependent.The half minimum inhibitory concentration(IC₅₀)of baicalin was calculated by the determination of toxicity valence as24.02μM,and the selectivity index SI=6.37.Western Blot was used to detect the expression of PRV US3 at different concentrations of baicalin6 h,9 h,12 h,and 24 h after PRV infection,and the results showed that baicalin significantly inhibited the expression of PRV US3,further proving that the antiviral activity of baicalin is dose-dependent.Studies have shown that baicalin has an inactivating effect on viruses.The incubation of PRV and baicalin（96μM）at 37°C for 3 hours reduced the toxicity of PRV from（1.38±0.6）×10⁸ TCID₅₀/m L to（4.27±0.56）×10⁷ TCID₅₀/m L compared with the DMSO control group,indicating that baicalin had a mild inactivation effect on PRV.This study further evaluated the effect of different dosing times on the anti-PRV effect of baicalin.First,PK-15 cells were treated with 96μM baicalin for 3 h or 6 h in advance,and then PRV was used to infect the cells.Compared with the DMSO control group,3 h or 6 h in advance treatment of cells could significantly inhibit the replication of PRV.In the delayed dosing experiment,the dosing time of baicalin was 0 h,1 h,3 h,6 h after infection,and the toxicity was determined 24 h after PRV infection,and the results showed that 0 h,1 h and 3 h after infection could significantly inhibit the replication of PRV,and the inhibitory effect of 1h and 3h after infection was significantly weaker than that of 0 h after infection,and baicalin had no inhibitory effect on PRV replication 6 h after infection.（2）In this study,the effect of carbazoles on PRV inhibition in vitro was preliminarily evaluated:the inhibitory effect and cytotoxicity of 18 carbazoles on PRV were detected,and the results showed that No.The inhibition efficiency of 4,No.10,and No.16 compounds on PRV was above 80%,which was significantly higher than that of the other 15 carbazole compounds.These three compounds（No.4,No.10,No.16）,half of the cytotoxic concentrations were 33.12μM,62.42μM,and 42.98μM,respectively.On this basis,We further evaluated the 50%inhibitory concentration and selection index of compounds No.4,No.10 and No.16 on PRV,IC50 are 9.86μM,11.08μM and 12.79μM,and SIs are 3.36,5.63 and 3.36,respectively,and the results showed that these three compounds could effectively inhibit the replication of PRV with good safety and potential for further development.In summary,this study shows that the plant extracts baicalin and carbazoles can effectively inhibit PRV replication in vitro,it was demonstrated that baicalin inhibits PRV replication by multiple mechanisms,both directly inactivating the virus and inhibiting viral replication by regulating host genes in the early stages of infection.This study provides updated information and clues for the screening and development of anti-PRV actives in plant extracts.In the future,it is necessary to evaluate the effectiveness of these two classes of drugs in animals.