Screen Of Herbs Against TGEV And Preliminary Study On The Mechanism Of Effect

[Objective]Transmissible gastroenteritis virus(TGEV) could cause pigs acute gastroenteritis,vomit,dehydration,high mortality in piglets within two weeks,which had lead to serious loss against swine industry.This experiment firstly built up TGEV infectious model in vitro,and then measured the safety concentration of Chinese medicines extracts prepared from antivirus herbs,and finally detected the antivirial effect of these herbs in order to provide theoretical basis for clinical application.[methods](1)The PK-15 cells were cultured till the monolayer was formed.And then the monolayer was inoculated with the TGEV while the growth state of cells was observed.The TGEV could be passaged blindly for many generations until the cytopathic effect (CPE) appeared.The TGEV products should be harvested after 3 times of freeze-thawing and stored at -70℃.According to the conservative domain of sM gene and 3′-UTR fragment of TGEV,two pairs of primers(P1/P2,P3/P4)were designed and synthesized. RT-PCR was used to detect the RNA of TGEV.In addition, 3′-UTR fragment was sequenced for homology alignment with that of other TGEV strains.The anti-TGEV antibody was made from immuined mice and the indirect immunofluorescence assay(IFA) was also applied for detection of TGEV. (2)The effective component of Chinese herbs was extracted by water-boiling method and the terminal concentration of herb was 100mg/mL.The extracts should be sterilized and stored at 4℃.The serial dilution of extracts with cell maintenance solution was prepared for detection of herb cytotoxicity.The interaction of herb extracts with cells should be maintained for 72h. Finally the maximum safe concentration of herbs was determined by observing the morphology of cells and the integrity of cell monolayer.(3) The extracts of herbs was doule-dilluted on the basis of the largest safety concentrion .The effect of anti-TGEV of herbs was studied in three ways which were adding medicine before adding virus,adding medicine after virus and adding medicines and virus simultaneously.The live cell quantity was measured by MTT method after herbs interacted with virus for 3d and the function of anti-TGEV of herbs was analysized by T-test.[Results](1)TGEV could cause obvious cytopathic effect (CPE) after four blind passages and characterized as roundout,detach,lysis,et al. while the morphologyl of control cells was normal.The RT-PCR assay of TGEV infected cells displayed two expected positive bands of size of 516bp and 346 bp.Sequence analysis of TGEV showed that the nucleotide sequence homology of 3'-UTR fragment shared 97%~99% with that of other TGEV strains.The specific green fluorescence could be observed in the TGEV infected PK-15 cells by IFA.These assays together approved the existence of TGEV.(2)According to observation of the morphology of cells,the extract of herbs with high concentrition(>25 mg/mL) could cause cell destroyed.In general, maximum safe concentration of herbs ranged from 6.25~12.5mg/mL.But that of astragalus(huangqi)was even about 50 mg/mL.(3)The MTT experiment approved that rhubarb(DaHuang),Paris (ZhongLou) and Loosestrife (JinQianCao)could inactivate the TGEV directly,and the therapeutic index(TI) was 8,8,16,respectively;Astragalus (HuangQi) and Scutellaria(HuangQin) could keep TGEV from the absorbtion to PK-15 cells,and TI was 8 and 16, respectively;but we have not found herbs which could inhibit the replication of TGEV.[Conclusions](1)TGEV could cause cytopathic effect through four blind passages and could be approved by RT-PCR,nucleotide sequence detection and IFA.(2)The largest safety concentriton of the extracts of herbs ranged from 6.25~12.5mg/mL on the PK-15 cells.(3) Astragalus (HuangQi) and Scutellaria (HuangQin) could inhibit the absorbtion of TGEV,and Rhubarb(DaHuang),Paris(ZhongLou) and Loosestrife(Jin- QianCao) could inactivate the TGEV directly, but no herbs found could inbibit the replication of TGEV.