Comparative Transcriptomics Of PEDV CV777 And HLJ Strains And The Antiviral Mechanism Of PTRIM7

Porcine epidemic diarrhoea(PED)is an acute and highly contagious intestinal disease caused by porcine epidemic diarrhoea virus(PEDV),which belongs to the Alphacoronavirus(α-Co V)in Coronaviridae and can cause disease in pigs of all ages,with the highest incidence in piglets.PED is characterised by vomiting,diarrhoea and dehydration of piglets,with mortality rates of up to100%,causing serious damage to the pig industry in China and huge economic losses to the global pig industry.There are several genotypes of PEDVs,and it becomes more and more complicated for the continuous recombinations and evolutions among different virus strains.Although vaccines for PED have been developed,clinical use has revealed low vaccine protection rates,and poor cross-protection against prevalent virus strains owning to the commercial vaccines were prepared with traditional virus strains,this made it difficult to prevent and control the infection with prevalent strains of PEDV.Therefore,studying the differences in infection and pathogenesis of different genotypes of PEDV has far-reaching implications for the prevention and control of PED.In this study,the PEDV HLJ strain was isolated and identified using cell culture techniques,and adaptive cultures were obtained in porcine small intestinal epithelial cells(IPEC-J2).The differences between the gene sequences of this strain and the conventional CV777 strain were analyzed bioinformatically after cloning and sequencing its entire genome.Subsequently,a comparative transcriptomic analysis of the IPEC-J2 cells infected with PEDV traditional and prevalent strains was carried out based on RNA-sequencing(RNA-seq)technology to provide a reference for unraveling the PEDV pathogenesis and host immune response.Based on the results of transcriptomic analysis,the role of porcine tripartite motif 7(p TRIM7)protein in PEDV infestation was investigated to provide a reference for revealing the antiviral mechanism of TRIM7.The effects of PEDV and various viral proteins on TRIM7 were investigated,and new innate immune suppression strategies for PEDV were identified,providing a reference for the immune escape of PEDV and the development of related drugs.The main studies are as follows:1.In this experiment,PEDV CV777 and HLJ strains were passaged on IPEC-J2 cells for fifteen consecutive generations.After 24 h of virus infection,the cells showed rounding,crinkling,fusion and shedding,and PEDV-specific green fluorescence was observed in the cell pulp by indirect immunofluorescence technique.The growth curves of CV777 and HLJ strains were further plotted,indicating that both strains of PEDV could obtain adaptive amplification on IPEC-J2 cells,and the titer of HLJ strain was higher than that of CV777 strain.2.In this study,the homology and phylogenetic analysis of the whole genome,S and ORF3 proteins of the HLJ strain and 28 representative strains revealed that the HLJ strain had the highest homology with the PEDV-SX strain and belonged to the GIb subgroup.Analysis of the difference sites in antigenic epitope revealed have five mutations in the core neutralization epitope(COE)domain of the HLJ strain,and recombination analysis revealed that the HLJ strain could be the parental strain in the production of the TW-yunlin-550 recombinant strain.Further analysis of the phosphorylation and glycosylation sites of HLJ and CV777 strains revealed that the S protein of HLJ strain lost two N-glycosylation sites at positions 57 and 1258,while a new glycosylation site appeared at position 112,and 14 phosphorylation sites were missing and 11 new phosphorylation sites were found in the S protein of HLJ strain,suggesting that this may be the reason for the different virulence of HLJ and CV777 strains.3.In this study,transcriptomic changes in IPEC-J2 cells infected with traditional and prevalent strains of PEDV were investigated using RNA sequencing technology.There were 744 and 258 differentially expressed genes(DEGs)identified in the CV777 and HLJ strain infection groups compared with the control group,respectively,with 201 DEGs between the CV777 and HLJ infection groups.Bioinformatics analysis revealed that the differential proteins were mainly involved in signal transduction,immune response and inflammatory response processes.Comparative analysis of the differential genes between the two virus strains revealed that antiviral innate immunity genes,inflammatory factors and chemokines showed different expression profiles,suggesting that this may be a factor contributing to the difference in pathogenicity between these two strains.4.In this study,the TRIM7 protein was found to be inhibited during infection by CV777 and HLJ strain for the first time.To investigate the mechanism of TRIM7 in viral infection,the gene sequence of TRIM7 was first cloned and bioinformatically studied.The TRIM7 gene encodes 510 amino acid residues and includes a large RBCC structural domain(aa 29-272)at the N-terminal end and a B30.2(PRY/SPRY)structural domain(aa 340-506)at the C-terminal end.Genetic evolutionary analysis showed that p TRIM7 belongs to the mammalian branch,has a maximum of96.6% homology with alpaca TRIM7.Relatively high expression of TRIM7 protein was detected by Western blot in kidney,liver and lymph nodes,while lower expression in alimentary tracts and muscle,suggesting that TRIM7 may affect the host metabolic and immune functions.5.In this study,the role of TRIM7 in inhibiting PEDV replication and reducing viral titers was confirmed by TRIM7 overexpression and si RNA silencing in IPEC-J2 cells,indicating that TRIM7 is a restriction factor for PEDV replication in host cells.Real-time quantitative PCR detected that overexpression of TRIM7 activated the expression of RIG-1,MAVS,TRAF6,TBK1,NF-κB,IFN-β,TNF-α,IL-6 and IL-8 genes,and silencing TRIM7 decreased the expression levels of the above-mentioned genes,suggesting that TRIM7 could be involved in the transactivation of innate immune signalling pathways.6.Host proteins interacting with TRIM7 were identified using immunoprecipitation combined with mass spectrometry,and Ras GTPase-activating protein binding protein 1(G3BP1),DEx D/H-box RNA decapping enzyme(DDX1)and DDX3 X were selected from these 104 interacting proteins for validation.Co-localisation between TRIM7 and G3BP1,DDX1 and DDX3 X was found with confocal microscopy,indicating a spatial interaction between TRIM7 and the 2 interested proteins.Using immunoprecipitation techniques,the interactions between TRIM7 and G3BP1,DDX1 and DDX3 X were further confirmed.Overexpression of G3BP1,DDX1 and DDX3 X in IPEC-J2 cells could inhibit PEDV replication and reduce viral titer,and G3BP1,DDX1 and DDX3X significantly activated the expression of IFN-β.In addition,TRIM7 could activate the transcription of G3BP1,DDX1 and DDX3 X,suggesting that TRIM7 could play an inhibitory role in PEDV replication by activating the transcription of G3BP1,DDX1 and DDX3 X and further regulating the expression of IFN-β.7.In this experiment,the effect of PEDV on the TRIM7 gene and protein was studied by different infection doses and times.The virus could promote TRIM7 with a trend of early activation followed by inhibition,which may have been influenced by viral replication.Eukaryotic expression plasmids for PEDV structural and non-structural proteins were subsequently constructed.PEDV NSP15,N and M proteins were screened by both real-time quantitative PCR and Western blot to inhibit TRIM7 gene transcription and protein expression,indicating that certain viral proteins were the main factor in the inhibition of TRIM7 expression,but immunoprecipitation experiments revealed that PEDV NSP15,N and M proteins did not interact with TRIM7.In summary,this study obtained adaptive culture of the HLJ strain of PEDV endemic strain in Heilongjiang on IPEC-J2 cells,providing more options for vaccine preparation of the PEDV endemic strain.And more references for the evolution and genetic characteristics of the PEDV endemic strain through bioinformatic analysis.Using comparative transcriptomics,we investigated the differential genetic changes in IPEC-J2 cells infected by PEDV CV777 and HLJ strains,and then explored the role of TRIM7 in the process of PEDV infestation and its mechanism,and identified that PEDV NSP15,N and M proteins could antagonize the transcription and protein expression of TRIM7,which provide further theoretical basis for the pathogenesis of PEDV and related drug development.