Isolation And Identification Of Porcine Transmissible Gastroenteritis Virus HB-FP Strain And Effects Of Autophagy On TGEV Proliration

Transmissible Gastroenteritis(TGE)is an acute,highly contagious intestinal infectious disease caused by the porcine Transmissible Gastroenteritis virus(TGEV),a member of the Coronavirus family Coronavirus genus.The main clinical symptoms of the disease are watery diarrhea,dehydration.The mortality rate of piglets under 2 weeks of age can reach 100%,which brings serious economic losses to the swine industry.At present,TGE is mainly prevented by vaccination,but the viruses that have prevailed in recent years are in a process of constantly changing virulence and biological characteristics,traditional vaccines have failed to protect against popular strains.In this paper,the TGEV epidemic strains in Hebei region of China were isolated and identified,and a stable cell-adapted strain was obtained;The genetic evolution of S and M genes of the isolate was analyzed and the effect of autophagy on TGEV replication were investigated.The results of this research not only provide an important theoretical basis for the research on the pathogenic mechanism of TGEV epidemic strains,but also have important significance in the prevention and control of TGE and the development of new vaccines.In this study,piglet feces and small intestine contents with diarrhea were collected from Hebei Province,and the positive materials detected by RT-PCR were inoculated into PK-15 cells for transmission.Typical cytopathy was observed in the 5th generation,the harvested asmples were RT-PCR tested for only TGEV positive.According to the Reed-Muench method,the 10th generation cytotoxic TCID50 was calculated to be 10-5.5/0.1mL.Indirect immunofluorescence assay further confirmed that the virus can specifically bind to the TGEV rabbit-derived whole virus polyclonal antibody after infecting cells,and specific immunofluorescence appears.these results verified that the isolated virus was transmissible gastroenteritis virus,and named TGEV HB-FP strain.The S,M gene of HB-FP strain was amplified by RT-PCR and analyzed along with the sequence alignment and phylogenetic trees of TGEV S,M gene sequences from other domestic and international.The results showed that there was no base deletion or insertion in the M gene,and the nucleotide homology between the isolate and the reference gene was higher than 95%,and the highest nucleotide homology with TGEV H16(FJ755618)was 99.9%,The amino acid homology is 99.2%.The results showed that the S gene of HB-FP strain had 94.9%～98.8%nucleotide homology and 65%～98%amino acid homology with other reference strains.The highest homology with TGEV JS2012(KT696544)was 98.8%in nucleotide and 98%in amino acid.Phylogenetic tree analysis showed that the HB-FP strain was on the same branch as the other reference strains except that it was not on a branch with the British TGEV strain(AF104420),among them,the TGEV JS2012(KT696544)strain in China,the TOY56-165(M94103)strain in the United States,the TGEV(AF298211)strain in Korea,and the isolate HB-FP strain have the closest relationship.The analysis of amino acid variation showed that there were 21 amino acid point mutations in the isolates and commonly used vaccine strains,including 7 point mutations in the epitope region of S protein,the effect of mutation of these antigen sites on the biological characteristics of TGEV remains to be further studied.In this study,TGEV HB-FP strains with different amounts of virus were used to infect PK-15 cells,and the expression levels of autophagy-associated protein were detected by Western blotting.It was confirmed that TGEV infection can cause PK-15 cell autophagy.UV inactivated virus can cause cell autophagy.Although the expression of LC3-Ⅱ/β-actin is increased compared with the control group,its ability to induce autophagy is significantly lower than that of the normal virus group,the results showed that the UV-inactivated TGEV did not completely lose the ability of inducing autophagy of PK-15 cells,but the replication process of virus could promote autophagy of host cells.To investigate the effect of autophagy on the proliferation of TGEV,it found that rapamycin can increase the titer of TGEV after treating PK-15 cells,while 3-MA can inhibit the replication of TGEV,indicating that autophagy can promote the replication of TGEV.After infecting PK-15 cells with TGEV HB-FP strain,the transcription of beclin-1 and ATG5 genes were significantly increased,the combination of virus proliferation curves confirmed that TGEV HB-FP strain had a positive effect on virus proliferation and cell autophagy after infection of PK-15 cells.The research results provide a new perspective for further exploring the interaction between TGEV HB-FP strain and host cells and the pathogenesis of TGEV.