Isolation And Identification Of Porcine Transmissible Gastroenteritis Virus HX Strain& Machnism Of PL Protein Antagnize The Signaling Pathway Of IFN-β

Transmissible gastroenteritis virus of swine(TGEV) is an enveloped virus and belongs to a member of groups of coronaviruses, which is one of the most important causative agents of enteric infection. The infection is associated with high morbidity in animals of all ages and with high mortality in suckling piglets. TGE can cause substantial economic losses in pig industry because of its wide host range.The samples were collected from a pig farm in Heilongjiang province with diarrhea, and then identified by RT-PCR. The N gene primers were designed by software, and the amplicon is 1100 bp long. Meanwhile, the primers of PRV, PRRSV and PEDV were employed by RT-PCR for specificity. The results showed that the diarrhea samples were contained TGEV as a result of the N gene sequence homology was 98.5%, compared with other TGEVs. PCR-positive viral samples were inoculated into PK-15 cells, which were grown as a monolayer in Dulbecco’s modified Eagle medium containing 10% fetal calf serumand 5 % CO2 in air. Viruses were passaged eight times and were harvested by three cycles of freezing and thawing. Cellular debris was removed by low speed centrifugation at 3,000 × g for 10 min, and the supernatant was aliquoted and stored at-80°C.PK-15 cells infected with TGEV were harvested by freezing and thawing three times. One m L of cell culture was centrifuged for 5 min at 800 × g. The supernatant was transferred into a new microfuge tube and centrifuged for 10 min at 13,400 × g. Then, the pellet was negatively stained with 2% phosphotungstic acid and analyzed on a transmission electron microscope. The full-length genome sequence of the TGEV-HX strain was deduced by combining the sequences of 10 overlapping c DNA fragments. The genome sequence of the TGEV-HX strain was 28,580 nucleotides(nt) long. The genes of TGEV-HX were arranged in the order 5 ’-UTR-ORF1a-ORF1b-S-ORF3a-ORF3b-E-N-ORF7-3’ UTR. The homology results suggested that TGEV-HX showed higher identity to strains SC-Y, WH-1, and Purdue, and less identity to TS, Miller 6, and H165. The TGEV-HX strain had a close relationship with the Purdue strain and is more distant evolutionarily from the Miller strains group and strain ISU-1.Innate immune response is the first line of host defense system for invading pathogen sensing and eliminating with minimal clinical consequences. Coronaviridae viruses(Co Vs), severe acute respiratory syndrome(SARS) and porcine epidemic diarrhea virus(PEDV), have developed diverse strategies to inhibit type Ⅰ interferon(IFN) and prolong their own survival. Moreover, transmissible gastroenteritis virus(TGEV) can induce robest the production of IFN-α, This study aims to reveal the relationship between inflammatory factors and TGEV infection. PK-15 cells were infected by TGEV-HX or inactive TGEV-HX. q Real-time PCR was carried out to detect the transcript level of cytokines(IFN-α1, IFN-β, IL6, IL12, TNF-α, IRF7, STAT1, ISG56 and Mx1). The results showed that the induction of IFN-α1, IFN-β, TNF-α, IRF7 and ISG56 was further blocked by TGEV-HX infection compared with inactive TGEV-HX. Both TGEV and inactive TGEV induced a significant increase of STAT1. In addition, TGEV can induce relatively more robust expression of IL12 and Mx1 in comparison to inactive TGEV-HX.Coronaviruses(Co V), such as human coronavirus NL63(HCo V-NL63), severe acute respiratory syndrome Co V(SARS-Co V), murine hepatitis virus(MHV) and porcine epidemic diarrhea virus(PEDV), encode papain-like(PL) proteases that inhibit Sendai virus(Se V)-induced interferon(IFN-β) production. Recently, the crystal structure of transmissible gastroenteritis virus(TGEV) PL1 has been revealed that it is similar to that of SARS-Co V PL2 pro, which may antagonize host innate immunity. We initially showed that TGEV PL1 encoded by the replicase gene blocked the activation of the IFN-β promoter and inhibited nuclear translocation of interferon regulatory factor 3(IRF3). The ability to antagonize IFN production was dependent on intact catalytic activity of PL1. Furthermore, TGEV PL1 exerted deubiquitinase(DUB) activity which strongly inhibited retinoic acid-induced gene I(RIG-1)- and stimulator of interferon gene(STING)-dependent IFN expression.The present study provides the complete genome sequence of a TGEV-HX strain from China. By comparing the S gene and protein with those of other TGEV strains, we have gained a further understanding of the genetic structure, diversity, and evolution of the TGEV-HX strain.