A Multi-omics Study Of Serum Exosomes In Infection With CHsx1401 Strain Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus(PRRSV)is one of the most damaging pathogens in pig breeding,which can cause reproductive disorders in sows,respiratory diseases in piglets,reduced growth performance and other diseases leading to pig death.Exosomes are small biological vesicles secreted and released by cells,which act as mediators of intercellular communication and play a unique role in virus infection,antigen presentation,and suppression/promotion of immune response.In this study,we used the PRRSV NADC30-like CHsx1401 strain to artificially infect 42-day-old piglets and isolated serum exosomes by QIAGEN exo Easy Maxi Kit,and used a multi-omics study to dissect the differences in mi RNAs,proteins and metabolites in serum exosomes of pigs before(the control group)and after(the treatment group)PRRSV infection from a multidimensional perspective,aiming to elucidate the differences in the expression of serum exosomes that regulate PRRSV infection.The aim was to elucidate the changes in transcriptome expression,protein and metabolite levels in exosomes regulating PRRSV infection,and to identify key potential functional molecules in exosomes related to immunomodulatory mechanisms after PRRSV infection.The results were shown as follows:(Ⅰ).Comparison of blood index differences in piglets infected with PRRSV,isolation and identification of exosomes.Blood routine test and blood biochemistry indexes were measured in PRRSVfree(0 day post-inoculation)and PRRSV-positive(7 days post-inoculation)piglets,and significant differences were found in LY,MPV,MO%,AST and LDH were significantly different before and after the piglets were infected with CHSX1401 strain.Exosomes were isolated from serum in piglets before and after infection using an exosome isolation kit based on membrane affinity technology,and their shape,size and characteristic proteins were detected by TEM,NAT and Western blot methods,respectively.The results showed that(1)the shape of porcine serum exosomes was in the shape of a teat or disc with a depression in the middle;(2)the particle size of exosomes was mainly concentrated around 72.25 nm with an average diameter of 76.22 nm;(3)the characteristic proteins CD9 and CD81 were highly expressed in the isolated exosome samples.In summary,the shape,particle size and protein characteristics were consistent with the exosome characterization,suggesting that the obtained serum extracts were exosomes.(Ⅱ).Mi RNA analysis of porcine serum exosomes.Based on high-throughput sequencing technology,a total of 305 mi RNAs were identified in the serum exosomes of piglets before and after infection,among which33 mi RNAs were significantly differentially expressed between the control and treatment groups(13 up-regulated and 20 down-regulated).Sequence conservation analysis of the CHsx1401 genome identified 8 conserved regions,among which a total of 16 differentially expressed mi RNAs were predicted to bind in the conserved region closest to the 3’ UTR of the CHsx1401 genome,including five mi RNAs that could bind to the CHsx1401 3’ UTR(ssc-mi R-34 c,ssc-mi R-375,ssc-mi R-378,sscmi R-486,ssc-mi R-6529).Further analysis revealed that the target genes of differentially expressed mi RNAs were widely involved in exosomal function-related and innate immunity-related signaling pathways,and 18 differentially expressed mi RNAs associated with PRRSV infection and immunity were screened as potential functional molecules involved in the regulation of PRRSV virus infection by exosomes.(Ⅲ).Quantitative proteomics of TMT labeling of porcine serum exosomes.Based on LC/MS-MS mass spectrometry,a total of 145 differentially expressed exosomal proteins in piglets before and after infection were identified in this study.The differential proteins were widely involved in complement and innate and adaptive immunity related pathways.Among them,the 14-3-3 protein family,the heat shock protein 90α family and complement factors play a central role in the serum-derived exosomal protein network as candidate proteins against PRRSV infection in exosomes.(Ⅳ).Untargeted metabolomics analysis of porcine serum exosomes.Based on UPLC-MS ultra-high performance liquid chromatography-mass spectrometry,a total of 343 differentially expressed metabolites were identified,of which 34 and 7 different metabolites were obtained in positive and negative ion modes respectively.Differential expressed metabolites are significantly enriched in signal pathways such as tryptophan metabolism,primary bile acid biosynthesis,pantothenic acid and coenzyme A biosynthesis.Further analysis found that the metabolites of Tryptophan,L-Kynurenine,2-Arachidonoyl glycerol,phosphatidyl choline(PC)and Sphingomyelin(SM)in serum secretion may be related to the immune process after PRRSV infection.ROC analysis results screened 15 potential biomarker metabolites that play a role in the process of PRRSV infection.In summary,we compared the changes in the expression of mi RNAs,proteins and metabolites in serum exosomes before and after infection with PRRSV NADC30-like CHsx1401 strain by multi-omics analysis,and used bioinformatics analysis to screen for potential functional molecules associated with exosome regulation of PRRSV infection process.This study provides a new perspective to further investigate the molecular mechanism of the immune response triggered by PRRSV virus in pigs and provides a theoretical basis for the use of exosomes in the prevention and control of PRRSV infection.