Transcriptome Analysis Of IPEC-J2 Cells During PEDV,PDCoV And SADS-CoV Co-infection And Antiviral Investigation Of TLR3-and INSIG1-mediated Signal Pathway

The newly emerged and re-emerged viral diarrheal diseases have resulted in substantial economic losses in the pig industry around the world.Porcine epidemic diarrhea(PED)characterized by vomiting,severe watery diarrhea,and dehydration in seronegative pigs at all ages,with high morbidity and mortality in suckling pigs.Porcine epidemic diarrhea virus(PEDV),the number one killer of neonatal piglets,is the causative agent of PED with an infection rate of 60% and a mortality rate of 100%;Followed by porcine deltacoronavirus(PDCoV),with the infection rate of as high as 30%,and the mortality rate of > 50%.SADS-CoV is the most newly discovered coronavirus,which has been first reported in 2017 in southern China and is considered to be an HKU2-related coronavirus with a bat-origin.The onset time is about 2-3 days later than PEDV and generally less severe in its clinical manifestations.China is the number one country for pig farming in the world,with an annual pig slaughter in recent years about700 million,but there are only limited systematic report on the molecular epidemiology of PEDV,PDCoV,SADS-CoV infection and its genotyping in south of China.PEDV and PDCoV co-infections are common in the field,and the pathogenesis of the co-infection is roughly clear.To address the interactions and mechanisms induced by PEDV,PDCoV and SADs-CoV co-infection,and Poly(I:C)antiviral potentials,the following experiments were performed::1.Prevalence and phylogenetic analysis of porcine diarrhea associated viruses in southern China from 2012 to 2019A total of 3,051 specimens sampled in 168 pig farms from five provinces,including Jiangxi,Zhejiang,Guangdong,Fujian,and Hunan province in Southern China from 2012 to 2019 were tested in this study.The previously established PCR protocols were used to test three major diarrhea-associated viruses,PEDV,PDCoV and SADS-CoV.The results indicated that variant PEDV was the dominant virus associated with severe diarrhea with a prevalence rate of 57.06%;PDCoV was the second dominant virus detected and the positive rate was 27.14%;In this study,SADS-CoV was only identified in samples collected from Fujian province,but not observed in any samples from other four province,i.e.,Jiangxi,Zhejiang,Guangdong,and Hunan provinces.The positive rate of SADS-CoV was 0.23% in diarrheal samples.From 2012 to 2019,the positive rate of PEDV has been maintained at a as high as xx%.In 2013,the positive rate of PEDV was the highest,reaching 62.13%,and in 2019 the positive rate of PEDV was 45.31%,the lowest annual rate among the samples tested.The positive rate of PDCoV was the highest in 2018,The lowest in 2012;and SADS-CoV was detected only in 68 samples collected in Fujian in2017,with a total positive rate of 0.23%.In terms of the mixed infections of the samples,PEDV vs PDCoV co-infection was the most frequently observed accounting for 17.33% of the samples tested.Among the PDCoV positive samples,the highest PEDV and PDCoV mixed infection rate was 70.97%,and the lowest was the 2014 sample,which was 30.57%.To elucidate the genetic characteristics of PEDVs circulating in Southern China during 2012 to 2018,the complete S1 genes of 11 representative strains of PEDV were sequenced,and analyzed.Phylogenetically,the S1 regions(aa 1?794)of the 11 strains of PEDV identified in this study and other 73 selected reference PEDV strains were divided into two genotypes(genotype I: GI and genotype II: GII).All of the 11 strains determined in this study,along with 3 previously reported strains of PEDV were clustered into the GII,and subgroup GIIa.Compared with the CV777 vaccine strain,the strains determined in this study had the same mutations positioned at A518 S,G521D,L522H/Y,S524 G,V528I,T550 S,S563F,G594 S,A605E,L612 F,F617L,K630 T,E633V,and I635 V.The S1 gene sequences of PDCoV strains were obtained from eight representative PDCoV positive samples.To analyze the molecular characterization and phylogenetic relationships among PDCoV isolates from different countries,the eight PDCoVs determined in this study and39 reference strains of PDCoV retrieved from Gen Bank were used.sequence alignment analysis indicated that all eight PDCoV strains determined in this study along with all Chinese strains(except HKU15–44 and AN-2004)and a strain identified in Thailand(TT?1115)had the same 3-nt(AAT)deletion between nt 19,473 and 19,477 in the S gene,leading to a deletion of deduced aa Asparagine(Asn or N)when compared with other strains.The SADS-CoV-CH-NDFJ-2018 S1 gene isolated in this study had a nucleotide homology ranging from 97.2% to 100% with 41 reference strains in Gen Bank,while amino acid homology of the samples analyzed was between 98% and 100%;It had the lowest homology with SADS-CoV-CH-FJWT-2018(MH615810),a strain of SADS-CoV identified in Fujian and the highest homology with SADS-CoV?GDLX/2019(MK651076),a strain of SADS-CoV determined in Guangdong province.2.Study on the Biological Characteristics of IPEC-J2 Cell Infected by PEDV/PDCoV/SADS-CoVIPEC-J2 cells were used for PEDV,PDCoV,SADS-CoV separated/co-infections as model.Statistical analysis presented that virus titer increased significantly during PEDV single infection,comparing with PEDV+PDCoV and PEDV+SADS-CoV co-infections,which indicated that co-infection inhibited/affected the proliferation of viruses.During co-infections,PEDV virus copies were significantly lower in PEDV+PDCoV group than that in PEDV+SADS-CoV group,this showed that PDCoV had a stronger effect on PEDV proliferation than SADS-CoV in PEDV-related co-infection.Meanwhile,PDCoV has better proliferative activity,the titer of PDCoV was significantly higher,when co-infected with PEDV or SADS-CoV,than that of PDCoV single infection.Similar cases were observed in SADS-CoV separated/co-infections with PEDV/PDCoV.IFA results showed that cells can be infected by multi-viruses simultaneously without rejection.3.Comparative genomic transcriptomics analysis of IPEC-J2 during mono-infection and co-infection of PEDV,PDCoV,and SADS-CoVTo reveal a comprehensive profile of the transcriptome changes that occur within the IPEC-J2 cells during mono-and co-infection by PEDV,PDCoV and SADS-CoV,whole transcriptome analysis was performed based on the RNA-seq technology.Initially,the expression profiles of IPEC-J2 under each individual mono-infection model of PEDV,PDCoV,and SADS-CoV were determined.Then,the landscapes of transcriptome of IPEC-J2 cells under different combination of co-infection status,including PEDV +SADS-CoV,PEDV + PDCoV,and PDCoV + SADS-CoV co-infection were examined and compared with each other and the counterpart of individual mono-infection.The results demonstrated that PEDV + SADS-CoV induced a stronger immune response in the early stage of infection than PEDV + PDCoV co-infection,while PEDV + PDCoV co-infection caused a stronger inflammatory immune response in the later stage.In PEDV +SADS-CoV combination of co-infection,bioinformatics analysis on differentially expressed genes revealed that PEDV + SADS-CoV differential genes were mainly concentrated in amide biosynthetic process,ATP biosynthetic process,and ATP metabolic process pathways,and the differential genes in PEDV + PDCoV were mainly focused on innate immune response,immune system process,and immune response.In comparison of PDCoV co-infection with mono-infection,both PDCoV +SADS-CoV and PDCoV + PEDV co-infection,the numbers of differential genes underwent a continued increase with the infection time elongation.Bioinformatics analysis of differentially expressed genes revealed that PDCoV + SADS-CoV had more significant changes in cell signaling pathways than PDCoV + PEDV,and clustered pathways were different.At 24 h post infection(hpi),the PDCoV + SADS-CoV differential gene enrichment was most significant for innate immune response,response to cytokine,response to tumor necrosis factor,etc.,while the PDCoV + PEDV differential gene enrichment was for immune system process,immune response,and innate immune response.In comparison of SADS-CoV co-infection and mono-infection,the numbers of differential genes in SADS-CoV+PDCoV co-infection group kept increased with the duration of infection extension,while in the SADS-CoV+PEDV co-infection group,the numbers of differential genes at 48 hpi declined.In comparison of SADS-CoV co-infection and mono-infection,the numbers of differential genes in SADS-CoV + PDCoV co-infection increased as the duration of infection increased,while in the SADS-CoV +PEDV co-infection group,the numbers of differential genes at 48 hpi Declined.Bioinformatics analysis of differentially expressed genes revealed that GO enrichment analysis of SADS-CoV mono-infection group and SADS-CoV + PDCoV co-infection group were compared at different time points: including 6 hpi and 24 hpi.The results indicated that there was no significant difference Pathway enrichment;at 48 hpi,281 differential genes were significantly enriched to cellular components,but only microtubule cytoskeleton,nucleus,microtubule organizing center and centrosome pathways were statistically significantly different.Comparison of GO enrichment analysis between SADS-CoV mono-infection group and SADS-CoV+ PEDV co-infection group: At 6 hpi,there were significant differences in 38 enrichment pathways,among which the pathways related to viral immune response were significantly different,including RSAD2,MX1,DDX58,MX2,STAT1,OAS1,STAT2,CD40,IRF1,CCL5,ISG20,etc.;and no significant enrichment pathway was observed at 24 hpi and 48 hpi.In summary,the differentially expressed genes of the mono-infection and co-infection groups were compared with one another,and the differential genes were found to be mainly involved in immune response,cytokine pathway,cell adhesion,energy metabolism,and apoptosis pathways.We reported for the first time the differential expression profiles of host genes of IPEC-J2 cells during PEDV,PDCoV,SADS-CoV mono-infected and co-infection.The findings will increase our knowledge in understanding the changes of host immune response mechanisms at the gene expression level under mono-infection and co-infection statuses.4.Effects of TLR3 signal pathway on PEDV,PDCoV,SADS-CoVToll-like receptors 3,which rapidly stimulates innate immune system and induces the generation of adaptive immunity through identifying pathogen related molecules,is a kind of pattern recognition receptors of innate immunity.If a cell's defense system detects a double-stranded pathogen RNA,they will initiate corresponding mechanisms to destroy it so that to prevent from infection.TLR3 induces the production of type I interferons and pro-inflammatory cytokines and inhibits virus replication.Previous studies performed on transcriptome enrichment analysis found that the TLR3 pathway was significantly up-regulated in both mono-infected and co-infected groups.Poly(I: C)was used to stimulate IPEC-J2 cells before and after virus infection.It was found that the effects of 12 hours before virus infection on virus replication was more significant;and the use of BX795 to inhibit TLR3 pathway activation could promote virus replication.In order to further determine if the host's immune response was caused by the activation of TLR3 due to viral infection,p CAGGS-TLR3 overexpression plasmid and p CDNA3.1-U6-TLR3 interference plasmid were transfected to IPEC-J2 cells,respectively.The results showed that overexpression of TLR3 inhibited virus replication and caused more intense interferon production,and conversely reduced activation of the TLR3 pathway to promote virus replication.5.Interactions of the structural proteins of PEDV,PDCoV,and SADS-CoV and INSIG1In the process of virus infection of host cells,in addition to using the virus' s own proteins to suppress the host's immune response to a certain extent,it can also regulate the synthesis of sterols during virus infection as a strategy to promote virus replication and virus budding in lipid rafts.Insulin-induced genes are newly discovered major factors that regulate the synthesis and metabolism of sterols in cells in recent years.Viruses rely on their cellular hosts to provide energy and components for successful replication.In order to investigate the effect of PEDV,PDCoV,and SADS-CoV on cholesterol metabolism,viral protein overexpression recombinant plasmids were transfected to IPEC-J2 cells to study the relationship between INSIG1 and PEDV,PDCoV,and SADS-CoV.It was found that the levels of INSIG1 m RNAs and proteins were significantly down-regulated during the later stages of PDCoV infection,while PEDV and SADS-CoV were significantly up-regulated,which raised a question what is the underlying mechanisms for this phenomenon? Is it an intrinsic difference between alpha-and delta-coronavirus? More in-depth research work is obviously needed in the future to address the mechanisms.Overexpression of INSIG1 could reduce virus copy number of diarrhea-associated viruses;knockdown of INSIG1 could significantly promote virus expression.Co-infection studies have found that PDCoV could enhance the proliferation of PEDV and SADS-CoV to a certain extent.Therefore,the proteins produced by PEDV,PDCoV,and SADS-CoV might inhibit the INSIG1 gene.Studies on overexpressing the structural and non-structural proteins of the three viruses found that the virus S protein were able to significantly inhibit the expression of INSIG1,while other non-structural genes ORF3,NS3,NS6,NS7,NS7 a,and NS7 b could induce a up-regulation of INSIG1.To further understand the reasons for the significant expression differences of the INSIG1 of IPEC-J2 cells infected by PEDV,SADS-CoV and PDCoV in the late stage of infection,three viruses were cultured with addition of different concentrations of glucose medium.The results indicated that the expression level of the INSIG1 gene depended on the glucose concentration.By measuring the levels of INSIG1,AMFR,SCAP,SREBF1,INSIG2,DHCR24,LSS,MSMO1,and OSBPL8 which are related to cholesterol metabolism in the PDCoV infection,it was found that INSIG1,AMFR,SCAP,and SREBF1,,significantly decreased,and oxysterol binding protein like protein was significantly increased.This study established a new connection for the interaction of PEDV,PDCoV and SADS-CoV,and the metabolism of cholesterol,and provided a basis for future research on the lipidomics during mono-and/or co-infection of PEDV,PDCoV and SADS-CoV.