Comparative Transcriptomics Analysis Of Primary And Immortalized PAM Infected With PRRSV

Porcine reproductive and respiratory syndrome(Porcine reproductive and respiratory syndrome,PRRS)is an important viral infectious disease caused by PRRS virus(PRRSV)that seriously harms the pig industry.Porcine alveolar macrophage(PAM)is the main target cell for PRRSV infection and the best cell model for studying PRRSV infection and immune regulation mechanisms in vitro.However,the primary PAM is difficult to obtain,and the PAM obtained from pigs of different breeds,different ages,and different individuals is very different,which is not conducted to the basic research of PRRSV.In recent years,several international laboratories have developed immortalized porcine alveolar macrophage cell lines,but due to the loss or mutation of some important genes(such as PRRSV receptors)in the process of immortalization,there are only a few immortalized porcine alveolar macrophages is susceptible to PRRSV.IPAM(Immortalized PAM)are an immortalized porcine alveolar macrophage cell line highly susceptible to PRRSV.In order to explore whether this cell line is suitable as a cell model for PRRSV infection and immune mechanism research in vitro,this study first compared the dynamics of virus proliferation after PRRSV infection with IPAM and primary PAM.It was found that in the early stage of infection,IPAM was more susceptible to PRRSV than primary PAM;in order to explain the reason of this phenomenon,comparative transcriptomics analysis was performed on PRRSV-infected PAM and IPAM.The specific research content and results are as follows: 1.Analysis of PRRSV infection kinetics in PAM and IPAMPRRSV(WUH3 strain)was inoculated into PAM and IPAM at high dose(10 MOI),and cell lysates were collected at different times after infection(6,9,12,15,18,21,24,and 36 h)to determine the virus TCID50,and draw a one-step proliferation curve.The results showed that in the early stage of infection(6-15 h),the PRRSV proliferation titers on IPAM were significantly higher than those in PAM,especially at 12 h after infection.Further indirect immunofluorescence experiments were used to compare the infection rate of PRRSV in the early stage of infection(6,9,12,15 h)on two types of cells,indicating that the infection kinetics of PRRSV in PAM and IPAM are different,and in the early stage of infection,IPAM is more susceptible to PRRSV than PAM.2.Differential expression analysis of PRRSV-infected PAM and IPAMBased on the analysis of PRRSV infection kinetics on PAM and IPAM,PAM and IPAM infected with PRRSV 12 hours were selected for RNA-Seq analysis.Using the PRRSV ORF7 gene sequence as a reference sequence,the subread software was used to count the genomic copy number of PRRSV,and compare the PRRSV proliferation ability in PAM and IPAM.The results showed that the genomic copy number of PRRSV in IPAM is about 2.5 times than that in PAM,which is consistent with the results of infection kinetics.Further analysis of the differentially expressed genes after PRRSV infection of PAM and IPAM found that compared with its uninfected control group(PAM,IPAM),PAM produced 1285 differentially expressed genes after PRRSV infection,of which 812 were up-regulated genes and down-regulated genes were 473;IPAM produced 4316 differentially expressed genes after PRRSV infection,including 2578 up-regulated genes and 1738 down-regulated genes.Principal component analysis of the differentially expressed genes of these two cells revealed that PRRSV infection had a greater impact on the gene expression pattern of IPAM.3.The difference in immune response of PAM and IPAM to PRRSV infectionKEGG enrichment analysis was performed on the differentially expressed genes of PAM and IPAM infected with PRRSV,and it was found that the shared differentially expressed genes were preferentially enriched in immune-related signaling pathways such as TNF,MAPK,and IL17,indicating that the two cells had similar immune responses to PRRSV infection.Further analyzing the specific differentially expressed genes of PRRSV infection with PAM compared with the IPAM,the results showed that the differentially expressed genes after PRRSV infection with PAM were preferentially enriched in immune response,cytokine receptors,MAPK,TNF and other immune-related signaling pathways;the differential expression genes produced by PRRSV infection with IPAM compared with PAM is preferentially enriched in metabolism-related signaling pathways such as vesicle transport,fatty acid metabolism,and TCA.The above results indicate that PAM infection with PRRSV causes a stronger immune response,while IPAM infection with PRRSV causes stronger cell physiology and metabolic changes related to virus invasion and virus proliferation.4.Analysis of potential reason of differences in PRRSV infection rates and immune responses in PAM and IPAMPrevious studies have confirmed that the cell cycle affects PRRSV proliferation,PRRSV proliferation in G0 / M phase is higher than that in S phase.Therefore,further analysis of the differentially expressed genes related to cell cycle.The results showed that key genes regulating cell cycle downregulated after IPAM infection with PRRSV,such as BUB3,BUB1 B,CDK2,CDC25 C,HDAC10,HDAC1 and CASP8;In DEG of PAM,these genes are mostly up-regulated.It is speculated that the down-regulated expression of cell cycle-related genes in IPAM may lead to cell cycle arrest at the G0 / M phase,thereby promoting PRRSV proliferation.SLA molecules are pig-derived MHC molecules and play a key role in the processes of antigen presentation,immune recognition,and immune response regulation.Therefore,the analysis of DEG related to antigen presentation revealed that the expression of SLA-DOA,SLA-DQB,and SLAMF8 was not detected in either the PRRSV-treated group or the untreated group in IPAM.At the same time,the expression abundance of SLA-DMA,SLA-DMB and SLA-5 is extremely low in IPAM;the expression abundance of IFI30 and PDIA3 in IPAM is also significantly lower than that of PAM.It is speculated that in IPAM,the deletion or low abundance expression of genes related to antigen presentation may cause the lower immune response of IPAM to than PAM after PRRSV infection.Innate immune signaling pathways play an important role in the process of viral infection.In order to further study the difference between PAM and IPAM responses to PRRSV,this study performed an IPA analysis of the DEG and compared the differences in immune-related signaling pathways after PRRSV infection with PAM and IPAM.The results show that Nod-like receptor(NLR),Toll-like receptor(TLR),m TOR,AMPK,and PI3K-Akt signaling pathways are activated differently in two cells infected with PRRSV,which may lead to the different proliferation of PRRSV in PAM and IPAM.