LXR Agonist GW3965 Inhibits Classical Swine Fever Virus Infection By Regulating Cholesterol Homeostasis

Classical swine fever(CSF)is a contact infectious disease caused by classical swine fever virus(CSFV),which has caused huge economic losses to China.Studies have shown that liver X receptor(LXR)agonist GW3965 can inhibit the infection of HIV,Newcastle disease virus,hepatitis B virus and other viruses by regulating cholesterol homeostasis.In order to understand the prevalence of CSFV in Shandong Province and the mechanism of action between LXR and CSFV.We studied and discussed the CSFV infection in Shandong and its interaction with LXR cholesterol metabolism.The specific results are as follows:1.Investigated the status of CSFV infection in parts of Shandong from 2017 to2019In this study,1687 suspected disease materials and 4886 serum samples collected in various regions of Shandong Province from 2017 to 2019 were tested for CSFV E2 antigen and antibodies,respectively.The results showed that the antigen-positive rates of CSFV in Shandong Province from 2017 to 2019 were 8.6%,13.1%,and 15.1%,respectively,and the antigen-positive rate showed an upward trend year by year.The CSFV antibody test results maintained a high level,and the positive rates were 84.3%,83%,92.7%.Further isolation and amplification of 5 CSFV genomes and E2 gene protein sequences from positive disease materials revealed that the clinical homology of the 5 clinical isolates and the 2.1d subtype CSFV genome was between 96.8%and 99.5%.The results showed that the five isolates had a higher affinity with the 2.1d subtype of CSFV.The mutations in the amino acid positions of the isolates were at positions 102(L→M)and 159(K→R).Position159(K→R)is located in the main antigenic region of E2 protein.This study analyzed the prevalence and variation of CSFV in pig herds in parts of Shandong from 2017 to 2019,and provided a reference for guiding the prevention and control of CSF in Shandong Province.2.An absolute fluorescence quantitative method for CSFV-E0 was establishedIn this study,reference to the sequence of classical swine fever virus strain in Gen Bank,design specific primers in the E0 conserved region,construct a recombinant plasmid of CSFV-E0 and use it as a standard,establish a real-time fluorescent quantitative PCR standard curve,and perform specificity,sensitivity,and Repeatability test.The results showed that the cycle threshold of the standard curve had a good linear relationship with the template concentration(y=-3.172x+37.672,R2=0.9977);high sensitivity,the minimum detection limit was 1×10~2copies/μL;strong specificity,and pig Circovirus type 2,porcine parvovirus,pseudorabies virus,and porcine reproductive and respiratory syndrome virus have no cross-reactivity;the repeatability is good,and the coefficients of variation within and between groups are less than 2%.The results show that the established real-time quantitative PCR method has the advantages of specificity,sensitivity,and good repeatability,which lays the foundation for the quantitative analysis and clinical diagnosis of CSFV.3.LXR agonist GW3965 inhibits swine fever virus infection by regulating cholesterol homeostasisIn this study,cells and viruses were treated differently,and the effects of GW3965 on CSFV were explored from the three stages of adsorption,infiltration,and replication.The results showed that there was no significant change in the RNA content of the control group when CSFV was adsorbed.The virus RNA content of the control group and the GW3965 treatment group decreased significantly when CSFV was inoculated,and showed a concentration-dependent GW3965,indicating that GW3965 can inhibit virus infection by affecting CSFV invasion.When CSFV was replicated,the GW3965 treatment group compared with the control group,the viral RNA content decreased significantly at 12h and24h,and the decrease was more obvious with the increase of time,indicating that GW3965can inhibit the virus infection by inhibiting the CSFV replication.Furthermore,q RT-PCR was used to detect the effects of GW3965 on the inflammatory factors IL-6,IL-8,and IL-1βafter CSFV infection.The results showed that GW3965 can reduce the expression of IL-1β,indicating that GW3965 can inhibit the inflammation caused by CSFV effect.In addition,GW3965 can increase LXRαexpression in ST cells and its downstream lipid metabolism pathway genes(including SREBP-1,PPAR-γ,ABCA1,and FASN).At the same time,CSFV can also stimulate the expression of LXR and its downstream genes SREBP-1,PPAR-γand FASN on ST cells,while the expression of ABCA1 decreased.Therefore,it is speculated that the inhibitory effect of GW3965 on CSFV is achieved by increasing the expression of ABCA1 and promoting cholesterol efflux.4.Mechanism of CSFV invasion and cell membrane cholesterolIn this study,the mechanism of cell membrane cholesterol in ST cells infected with classical swine fever virus(CSFV)was explored.It was found that pretreatment with cholesterol extractant methyl-β-cyclodextrin to eliminate cell membrane cholesterol can inhibit CSFV invasion and inhibit The effect is dose-dependent;CSFV infection can be partially restored after exogenous cholesterol supplementation.Further monitoring the changes of ABCA1 and cholesterol content in ST cells infected with CSFV,the results showed that with the increase of time within 36 hours of infection,the content of ABCA1decreased and the cholesterol content increased.This shows that CSFV infection reduces the expression of ABCA1,leads to the accumulation of intracellular cholesterol and the formation of lipid rafts,which is conducive to CSFV invasion.This study provides new ideas for CSFV-targeted antiviral mechanisms.