Study On The Infection Characteristics Of TGEV And The Immunological Function Of IFN-? In The Interaction Of TGEV And Host Cells

Transmissible gastroenteritis(TGE)is an enteric disease of pigs causedby TGE virus(TGEV).The significant clinical symptoms of this disease are severe diarrhea,vomiting,dehydration,etc.Pigs of different ages are all susceptible,and it is highly lethal to newborn pialets,especially to the ages of piglets within two weeks,it can be up to 100%.Current the genome sequence,structure and main function of the protein of TGEV have more in-depth understanding,but the immune pathogenesis of TGE is still not clear,which hinder the development of new vaccines and specific drugs for TGE.Therefore,the main emphasis of the prevention and control work is to investigate the immune pathogenesis.In this paper,the infectious characteristics of TGEV on swine testicular(ST)cells and mature dendritic cells(m DC)were investigated..And swine signal transducer and activator of transcription(STAT1)gene was amplified,cloned and sequenced.The STAT1 protein was expressed in ST cells,and the expressing level of STAT1 was analyzed after TGEV infected host cells.The antiviral effect of IFN-? on TGEV replication in vitro was also studied,and the expression of IFN-? and IFN-signaling relating antiviral genes were also analyzed after TGEV infected ST cells and DC.The experimental contents are as follows:1.The peripheral blood mononuclear cell(PBMC)was isolated from healthy pigs.DC was initial separated using adherent method,rh GM-CSF and rh IL-4 were added to stimulate the DC to maturate,and then the morphology of mature DC was observed using the electron microscope.ST cells and m DC were infected with TGEV,and then cells and cell culture supernatants were collected at different times of post-inoculation(pi),the virus titer in the supernatant was test by TCID50 and the virus growth curve was drawed.The viral load of TGEV in the cells was detected by real-time PCR.The results showed TGEV can replicate in ST and m DC,and they have he same proliferation trend,which began to propagate after 3hpi,and up to the peak at 48 hpi,then began to decline,but TGEV virus titer in ST cells was far higher than that in DC.2.Primers were designed and synthesized according to the sequence of STAT1 in NCBI.RNA was extracted from ST cells,STAT1 gene was amplified,sequenced,and the evolution of swine STAT1 gene was analyzed.Then the STAT1 gene were subcloned into the eukaryotic expression plasmid p EGFP-N1 to obtained the p EGFP-STAT1.The recombinant plasmid was transfect-ed into ST cells with lipofectamine 2000,the expression of STAT1 protein was detected by Western blot.The STAT1 m RNA expressing level in ST cells after TGEV infection was detected by fluorescence quantitative PCR method.The results showed that the swine STAT1 gene was determined to be 2 274 nucleotides long.The STAT1 gene shared 99.9% to 100% nucleotide identities with other swine STAT1 sequences,and it shared 85.7% to 92.8% nucleotide identities with the STAT1 genes of other species available in Gen Bank.Western blot analysis indicated the target protein,indicating p EGFP-STAT1 was successfully expressed in ST cells.The expression of STAT1 gene in ST cells after TGEV infection began to increase at 12 hpi,and reached the peak at 24 hpi.3.In order to understand the inhibition of TGEV replication by IFN-? treatment,we used different concentrations of IFN-?(10 000,1 000,100,10,1 and 0 IU/m L)pretreated ST cells for 12 h,and then TGEV infected ST cells,when positive control cells showed obvious cytopathic effect,the cells of the IFN-a treated groups were collected,and RNA was extracted.TGEV titer of each group was detected by fluorescence quantitative PCR.The results showed that the content of TGEV in ST cells decreased with the increase of IFN-a concentration,in the 10 000 and 1 000 IU/m L groups,the IFN-a can completely inhibite the TGEV replication.It indicated that IFN-a strongly inhibited TGEV replication in vitro.4.To study the influence of TGEV infection on the IFN-a secreation and the IFN-a relating genes expression in the host cells,ST cells and DC were infected with TGEV,cells were collected at 3,6,12,24 and 48 hpi,the m RNA expression levels of IFN-? and IFN-? signaling pathway relating genes were tested by fluorescence quantitative PCR.The results showed that the TGEV infection of ST cells and DC can induce a large number of IFN-? expression,and DC showed high expression of IFN-a as early as 3 hpi.This displayed that DC plays an important role in the innate immune response against TGEV infection.In addition,TGEV infected DC and ST cells can increase the expression of antiviral cytokines in IFN-signaling pathway.Thisshowed that IFN-? produced during TGEV infection exerted its antiviral activity.