Antiviral Activity And Mechanism Of Resveratrol Against Pseudorabies Virus

Resveratrol(Res),a non-flavonoid polyphenol compound,widely exists in several higher plants,has been reported to have antiviral activity against herpes viruses.Pseudorabies virus(PRV),a herpes virus,is one of the most important pathogens of swine,resulting in devastating disease and economic losses worldwide.Nevertheless,there are currently no antiviral drugs available for PRV infection.In this study,Res was tested for anti-PRV activity,and the antiviral mechanism of Res aginst PRV was investigated.Furthermore,the antiviral activity of Res against PRV was investigated in piglets which were infected with Virulent Pseudorabies Virus.The main research contents and results are as follows:1.Antiviral activity of Res aginst PRV in vitro.CCK-8 assay and virus titer assay were used to evaluate the anti-PRV activity of Res in vitro.Res significantly(p <0.01)increased the cell viability of PRV infected PK-15 cells,and it significantly(p <0.01)inhibited the replication PRV in PK-15 cells.The half-toxic concentration of Res on PK-15 cells is more than 60 ?g / m L,and the half-inhibitory concentration of Res on PRV is 3.92± 0.08?g / m L,resulting a therapeutic index of more than 15.31 ± 0.33,which are highly effective and low-toxic compounds.These results demonstrate that Res has good anti-PRV activity in vitro.2.Anti-PRV mode of action of Res.Primers and probe were designed for targeting PRV g B gene.FQ-PCR was used to investigate the anti-PRV effect of Res on different modes of action and different dosing times.The results showed that a plasmid with PRV g B gene fragment was successfully cloned,and a standard curve,with a good linear relationship(R2 = 0.996)and high amplification efficiency(95.6%),was established by using the constructed plasmid.The anti-PRV activity of Res was not attributed to direct inactivation or inhibition of virus attachment to the host cells or protection cells free of PRV infection,but to the inhibition of viral replication in PK-15 cells.Res was added at 0h,1 h,2 h,4 h,and 8 h post infection to investigate the anti-PRV effect of different dosing times.The results showed that the virus titers of the treatment groups were lower than the untreatment group during 8 h post infection,and the anti-PRV activity of Res was limited to the first 8 h post infection.These reults demonstrate that Res exhibits anti-PRV activity by inhibiting virus replication in host cells,and its PRV inhibitory effect is limited to the early stage of PRV infecttion.3.The effect of Res on the transcription of PRV genes and cytokine genes.SYBR Green fluorescent quantitative PCR was used to determine the expression levels of internal reference gene(?-actin),immediate early gene(IE180),early genes(EP0,US1 and UL54),genes necessary for viral DNA synthesis(UL5,UL8,UL9,UL29,UL30,UL42 and UL52),and cytokine genes.The results showed that Res inhibited all of the detected PRV immediate early gene(IE180),early genes(EP0,US1,and UL54)and genes necessary for viral DNA synthesis(UL5,UL8,UL9,UL29,UL30,and UL42).Moreover,Res showed inhibitory effect on the transcription of IL-1?,IL-12p35,IFN-?,IFN-?,TNF-? and TNF-?and chemokine G,which were induced by PRV infection.These results demonstrate that Res has extensive inhibitory effect on the transcription of PRV genes and PK-15 cell cytokine genes that induced by PRV infection.4.Effects of Res on NF-?B and ERK1/2 cell signalling pathways of PRV-infected PK-15 cells.Western blot assay was used to investigate the effect of Res on ERK1/2 and NF-?B cell signalling pathways of PRV-infected PK-15 cells during 12 hpi,and a immunofluorescence assay was used to investigate the effects of Res on ERK1/2 and NF-?B cell signalling pathways related proteins levels and distribution.The results showed that PRV infection activated ERK1/2 and NF-?B cell signaling pathways of PK-15 cells,and Res showed no influcence on the activity of ERK1/2 cell signaling pathway of PRV-infected PK-15 cells.However,Res showed significant(p <0.05)inhibitory effect on the activation of NF-?B cell signaling pathway induced by PRV infection.The inhibitory effect is due to inhibit the degradation of I?B? and transfer of Rel A from the cytoplasm to the nucleus.Furthermore,the results of immunofluorescence assay confirmed the results of Western blot assay.These results demonstrate that Res is a potent inhibitor of both NF-?B activation and NF-?B-dependent gene expression through its ability to inhibit I?B kinase activity,which is the key regulator in NF-?B activation.Thus,the inhibitory effect of Res on PRV-induced cell death and gene expression may be due to its ability to inhibit the degradation of I?B kinase.5.Antiviral Effect of Resveratrol in Piglets Infected with Pseudorabies Virus.A piglet Aujeszky's disease model was established by nasal instillation of PRV.Different doses of Res were added to the feed for one week before infection.After the infection,the PRV-infected piglets were treated with different doses of resveratrol for 3 weeks.Clinical symptoms,survival rate,viral load,histopathological,and levels of serum cytokines were used to evaluate the anti-PRV activity of Res in piglets.The results show that Res can effectively reduce the mortality and increase the growth performance of PRV-infected piglets.After Res treatment,the viral loads significantly(p < 0.01)decreased.Pathological symptoms,particularly inflammation in the brain caused by PRV infection,were significantly(p < 0.01)relieved by the effects of Res.In Res-treated groups,higher levels of cytokines in serum,including interferon gama,interleukin 12,tumor necrosis factor-alpha and interferon alpha were observed at 7 days post infection.These results indicated that Res possesses potent inhibitory activity against PRV-infection through inhibiting viral reproduction,alleviating PRV-induced inflammation and enhancing animal immunity,suggesting that Res is expected to be a new alternative control measure for PRV infection.In summary,Res showed potent antiviral activity against PRV both in vitro and in vivo,and its mechanism of inhibiting PRV is due to two aspects: First,Res inhibits the degradation of I?B? induced by PRV,thereby inhibiting the activation of NF-?B cell signaling pathways,resulting in effectively inhibiting viral gene transcription,protein and DNA synthesis,and virus particle generation;second,Res reduces the inflammation caused by viral infection and increase the levels of antiviral cytokines.