The effect of matrine and glycyrrhizic acid on porcine reproductive and respiratory syndrome virus in Vitro and in vivo

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is endemic worldwide, seriously affecting the development of the pig industry, but vaccines have limited protective effects against PRRSV transmission. The aim of this study was to identify potential anti-PRRSV drugs. We examined the cytotoxicity of seven compounds formulated based on the mass ratio of glycyrrhizic acid to matrine and calculated their inhibition rates against PRRSV in vitro. The results showed that the seven compounds all had direct killing and therapeutic effects on PRRSV, and the compounds inhibited PRRSV replication in a time- and dose-dependent manner. The compound with the strongest anti-PRRSV effect was selected for subsequent in vivo experiments. Pigs were divided into a control group and a medication group for the in vivo evaluation. The results showed that pigs treated with the 4:1 compound had 100% morbidity after PRRSV challenge, and the mortality rate reached 75% on the 8th day of the virus challenge. These results suggest that this compound has no practical anti-PRRSV effect in vivo and can actually accelerate the death of infected pigs. Next, we further analyzed the pigs that exhibited semiprotective effects following vaccination with the compound to determine whether the compound can synergize with the vaccine in vivo. The results indicated that pigs treated with the compound had higher mortality rates and more severe clinical reactions after PRRSV infection (p < 0.05). The levels of proinflammatory cytokines (IL-6, IL-8, IL-1β, IFN-γ, and TNF-α) were significantly greater in the compound-treated pigs than in the positive control-treated pigs (p < 0.05), and there was no synergistic enhancement with the live attenuated PRRSV vaccine (p < 0.05). The compound enhanced the inflammatory response, prompted the body to produce excessive levels of inflammatory cytokines and caused body damage, preventing a therapeutic effect. In conclusion, the present study revealed that the in vitro effectiveness of these agents does not indicate that they are effective in vivo or useful for developing anti-PRRSV drugs. Our findings also showed that, to identify effective anti-PRRSV drugs, comprehensive drug screening is needed, for compounds with solid anti-inflammatory effects both in vitro and in vivo. Our study may aid in the development of new anti-PRRSV drugs.

Keywords: Antiviral activity against PRRSV; Glycyrrhizic acid; Matrine; Porcine reproductive and respiratory syndrome virus.

Fig. 1

Safe concentrations of the monomeric and compound drugs in MARC-145 cells. (A) Chemical structure of glycyrrhizic acid; (B) chemical structure of matrine; (C) TC₀ and TC₅₀ of matrine and glycyrrhizic acid monomers and compound drugs. Note: G:M indicates the mass ratio of glycyrrhizic acid to matrine; the abbreviation for ribavirin is R. A 4:1 ratio of glycyrrhizic acid to matrine had the strongest anti-PRRSV effects

Fig. 2

The antiviral effect of glycyrrhizic acid and matrine compounds in vivo. (A) Detailed grouping and challenge information for the time-of-addition assay; (B) Preventive effects of drugs on PRRSV; (C) Direct killing effect of drugs on PRRSV; (D) Therapeutic effects of drugs on PRRSV

Fig. 3

The effects of the 4:1 compound at different concentrations and different time points on proliferation

Fig. 4

Glycyrrhizic acid and matrine (4:1) inhibited the PRRSV-N protein expression. A. Nuclear staining; B. PRRSV-N protein staining; C. nuclear and PRRSV-N protein staining. MARC-145 cells were treated with G: M (4:1) for 72 h. Ribavirin was used as a positive control. The cells were examined by laser confocal microscopy. Bars, 100 μm

Fig. 5

Clinical symptoms in pigs from the in vivo experiments. (A) Grouping, morbidity and death rates obtained from the in vivo experiment; (B) Temperature of the pigs; (C) Changes in the antibody against the PRRSV N protein in the pigs. The data are from three independent experiments. The error bars denote standard errors of the means

Fig. 6

Determination of the synergistic immune effect of the compound drug vaccine. (A) Experimental groupings: immune drug group (IM), immunized control group (IMC), nonimmunized drug group (NIM), and nonimmunized control group (NIMC); (B) Antibody level of PRRSV N-protein in pigs; (C) Levels of IFN-α; (D) Levels of IL-1β; (E) Levels of IL-6; (F) Levels of TNF-α; (G) Levels of IL-8