Isolation,Identification And Genome Analysis Of A Chinese Field Strain Of TGEV

TGEV is the etiological agent of transmissible gastroenteritis(TGE),which can cause vomit,severe diarrhea and dehydration with high morbidity in pigs of all ages,as well as high mortality in suckling piglets with in two weeks,nearly 100%.TGE was first reported in the United States in 1946 and distributed wordwide after that,which usually outbreaks in cold seasons and cause significant economic losses in swine-raising farms.In this study,The TGEV AHHF strain was isolated from small intestines contents of TGE suspected piglets using PK15 cells,with 0.3% trypsin in the culture medium.This TGEV strain can cause typical cytopathic effect(CPE)and multiply stably in PK 15 cells.In order to confirm the virulence of TGEV-AHHF strain,pathogenicity analysis was tested on 3-day healthy newborn piglets.Twelve TGEV,PORV,PEDV-free piglets from one brood were selected and randomly divided into two groups,the test group and the control group.Each piglet in the test group was challenged with 1×106.12 TCID50 TGEV-AHHF strain,piglets of the control group were challenged with 10 m L DMEM per pig.Clinical signs were observed per 3h.The result show that piglets in test group begain to vomit and diarrhea at 48 h post challenging.No clinical signs were observed in the control group.Pathological examination show that small intestines were filled with yellow intestinal fluids and thin,translucent walls were observed in test group.Histological changes show that many mucosal epithelial cells were degenerate,necrotic and lysed and severe villous atrophy were observed in jejunum and ileum compared to the negative control.The result of quantitative Real-time PCR show that jejunum had the highest viral load followed by ileum,caecum,colon,rectum,stomach and lung.Our result indicated that TGEV-AHHF strain was a vilent strain which can cause typical Clinical signs,pathological and histological changes of TGE disease.The complete genome sequence of the TGEV-AHHF strain was deduced by RT-PCR,3?RACE and 5?RACE methods.A total of 28580 nucleotides were determined for TGEV-AHHF strain,encompassing the 5?non-translated region(nt 1-314),ORFs 1ab(nt 315-20,368),S(nt 20,365-24,711),3a(nt 24830-25,045),3b(nt 25,139-25,873),E(nt 25,860-26,108),M(nt 26,119-26,907),N(nt 26,920-28,068),7(nt 28,074-28,310)and the 3?non-translated region(nt 28,311-28,614).Percentage of nucleotide identity of TGEV-AHHF genome compared with others show that it shared high identity with WH-1 and SHXB strain.Phylogenetic analysis based on the complete genome sequences of the strain AHHF,other TGEV reference strains,and strain PRCV-ISU-1 show that AHHF Strain was more closely related to purdue group than to Miller group.Recombinant analysis showed that there were two potential recombination regions in the ORF1 a and S gene of the TGEV-AHHF strain.It may be the result of recombination between the purdue cluster and the Miller cluster.Isolation,genome property and pathogenicity analysis of TGEV strain will not only contribute to the basic reseach of TGEV,but also assist in developing biological products,the diagnosis and controlling of TGEV field strain.