Establishment Of TGEV Inhibitor Screening Method And Preliminary Determination Of Its Mechanism

Transmissible Gastroenteritis Virus(TGEV)is a type of coronavirus that infects pigs of all ages and has a mortality rate of 100%in piglets under 2 weeks old and 25-30%in piglets ages ranging from 2 to 6 months.Its symptoms include vomiting,diarrhea,weight loss and severe dehydration.Due to its rapid spread via fecal-oral route within farms,this virus causes huge economical loss in the pig farming industry.Presently,there are no specific antiviral treatments targeting TGEV,thus screening and designing a chemical drug against TGEV has broad marketing and clinical application values.This study is based on the screening of TGEV inhibitors using a Drug Library composed of 978 different compounds.From this library,6 compounds were found to have a potential inhibitory effect out of which 2 compounds were discovered to have a greater inhibitory effect on the TGEV virus.Both compounds could present as potential candidates for the treatment of TGEV.The main results are as follows:1.Establishing a high-throughput screening model for TGEVThe cell model for screening the inhibition of TGEV was based on the cell seeding density,detection time,viral infection dose,DMSO concentration on cell viability.PK-15 cells were passaged,and the density of cells was adjusted to the cell growth of 5×104 cell/mL,seeded in 96-well plates for 14h.The wells were treated with compounds of different concentrations,incubated for Ih then infected with the minimum infection dose of the virus 0.1 MOI.Cell viability was detected after 120h.The concentration of DMSO used during the experiments was equal or less than 2%.The primary and secondary screening narrowed down the selection to 6 compounds and by the third screening;the selection was narrowed down to 2 compounds.2.Verification of compounds inhibitory effect on TGEVThe 6 compounds were specifically tested on PK-15 cells,the inhibition ratio of the compounds at different concentrations against TGEV was detected,2 compounds showed an inhibition ratio higher than 60%,which are Apatinib Mesylate and Acitretin.CC50 values were detected through cell cytotoxicity assay,moreover experiments such as Western Blot and IFA were carried out to test the protein expression of TGEV in cells,each compound was tested to find the most efficient concentration needed in future application.These results showed that TGEV-N protein expression decreased when the concentration of the compounds increased.Therefore these 2 compounds all exhibit significant antiviral effects.3.Identifying the compounds initial inhibitory mechanism on TGEVThe mechanism of 2 compounds inhibiting TGEV was discovered by analyzing the effect of the drugs on the virus on 4 stages:cell pre-treatment,pre-absorption and absorption.The supernatant was then measured at different concentrations of virus titer;the results determined that Apatinib Mesylate at a concentration of 25 ?M is most effective on the virus attachment phase whereas Acitretin at a concentration of 50?M is most effective on the pre-absorption phase and has some effect on the absorption phase.4.Identifying the effect of Apatinib Mesylate on TGEVPrevious studies have shown that Apatinib Mesylate through the vascular endothelial growth factor receptor-2 VEGEFR2 can modulate the expression of Aminopeptidase N/CD13.Western Blot was applied on cells treated with Apatinib Mesylate at different concentrations and TGEV to detect the concentration of Aminopeptidase N/CD13 protein expression,these results showed that Aminopeptidase N/CD13 protein expression decreased when the concentration of the compound increased.Identifying compounds that inhibit TGEV and their initial role on the stages of the viral mechanism has helped lay the basis for the development of therapeutics and provides a reference for further research on TGEV's pathogenic mechanism.