Identify Differences In Immune Response Between Tongcheng And Large White Pigs Artificially Infected With PRRSV By Transcriptome Sequencing And Proteome Profiling

Porcine Reproductive and Respiratory Syndrome(PRRS),commonly known as "blue-ear pig disease",is caused by PRRS Virus(PRRSV).PRRS causes severe respiratory disorder in piglets,reproductive failure in sows and declining semen quality in boars.Due to its high genetic variability and PRRSV-induce immunosuppression,PRRS has brought huge economic losses to the pig industry,which has made PRRS become one of the most important infectious diseases in pig industry.However,during the outbreak of PRRS in 2006,none of the Tongcheng pigs(TC pigs,an indigenous Chinese local breed located in Tongcheng County,China)in Tongcheng Breeding Farm and a commercial farm was found dead caused by PRRSV infection,which suggested that TC pigs may have stronger resistance against PRRSV than other pig breeds.To confirm this hypothesis,5-week-old TC and Large White(LW)pigs were given intramuscular injection of PRRSV Wu H3 strain.The clinical symptoms,blood routine index and serum cytokines were monitored and compared in this study.Immune response differences between two breeds were studied using transcriptome sequencing performed on porcine alveolar macrophages(PAMs;the main target cells of PRRSV),testis(hosts‘ reproductive organs),inguinal lymph nodes(ILN)and spleen(main peripheral immune organs).Futhermore,i TRAQ of ILN was performed to confirm transcriptome analysis.This study intends to investigate the immune response differences between TC and LW pigs against PRRSV and lay the foundation for the genetic mechanism of PRRS resistance in TC pigs.The main findings are as follows:1.The analysis of the clinical symptoms,blood routine index and serum cytokines level of TC and LW pigs artificially infected with PRRSVIn this study,twelve 5-week-old male TC and LW piglets were intramuscularly challenged with PRRSV Wu H3 or RPMI-1640 medium,respectively.The clinical symptoms and the change of blood routine index and serum cytokines were monitored and compared.The results showed that TC pigs in infected group had a less severe clinical symptoms and lung lesion.Even thoughthe feed intake reduced,all the individuals can eat and drink on their own.The serum virus proliferation peaked at 5 days post challenge(dpc)and the highest viral load was only 0.4 times than that in LW pigs.The blood routine index did not change too much and IFN-? in serum increased to 67 pg/m L at 5-7 dpc.However,the LW pigs showed severe respiratory problems,ataxia and reluctance to drink and eat,and one pig died in the morning at 7 dpc.The serum virus proliferation peaked at 3 dpc,the erythrocyte count kept on rising significantly,but white blood cell count,leukocyte count and platelet count decreased significantly.These may have a relationship to the serious hemorrhage in multiple organs of LW pigs.In addition,the IFN-? was not detected from the serum of the LW pigs,this might be caused by high level of IL-10 and IL-12p40.2.The transcriptome analysis of the PAMs,ILN,spleen and testis of TC and LW pigs artificially infected with PRRSVAll the piglets were euthanised at 7 dpc and PAMs,ILN,spleen and testis of 12 piglets(3 individuals in each group were chosen randomly)were collected for histological analysis,transcriptome sequencing or proteome profiling.The main results are as follows:(1)The transcriptome sequencing of PAMs acquired an individual 11G-16 G data,and 686 differentially expressed genes(DEGs)between the control groups,804 DEGs between the infected groups,1806 DEGs in TC pigs after infection,and 2155 DEGs in LW pigs after infection.The DEGs of TC pigs were enriched in phagosome,leukocyte infiltration and apoptosis.The DEGs of LW pigs were enriched in S1 P,G protein and PI3K-Akt and LW pigs had more down-regulated DEGs.These data suggest a higher level of MHC molecular in TC pigs might be beneficial to antigen presentation,promoting the leukocyte moving to the infection sites and causing immune response.In addition,apoptosis was suppressed to help cell survive and antigen presentation,so that the host could clear the virus quickly.However,a stronger suppression of phagosome formation signaling was observed in LW pigs,which may reduce their ability to present the antigen.Also,the down-regulated S1 P and G protein signaling might suppress the downstream immune response by suppressing PI3K-Akt pathway.(2)The detection of viral load and testosterone level identified that TC pigs had a 30 times lower viral load in testis.The histopathological section showed that the development stage of testis in TC pigs was much higher than LW pigs.It reminded the maturation of the blood-testis barrier in TC pigs was earlier than LW pigs and it might help TC pigs show resistance to PRRSV.The testosterone level of TC pigs was more than 3.74 ng/m L which was much higher LW pigs.The transcriptome sequencing of testis acquired the individual 16-23 G data,6315 DEGs between the control groups,6777 DEGs between the infected groups,739 DEGs in TC pigs after infection,and 1493 DEGs in LW pigs after infection.Approximately 2/3 DEGs between the control groups had a higher expression in TC pigs and they mainly enriched in spermatogenesis pathways including the development and synthesis.TC pigs had less DEGs after infection and enriched in less pathways.It might be the immune privilege protected TC pigs.However,TNF,JAK-STAT,TLRs,NF-?B,T cells and B cells signaling and the interleukins were commonly up-regulated in LW pigs.(3)The viral load analysis and histopathological section of ILN and spleen showed that TC pigs had lower viral load and LW pigs had a higher rate of necrotic cells.The transcriptome sequencing had acquired 464 DEGs between ILN among the control groups,2909 DEGs between spleen among the control groups;3802 DEGs in ILN of TC pigs after infection,2532 DEGs in ILN of LW pigs after infection;1814 DEGs in spleen of TC pigs after infection,2021 DEGs in spleen of LW pigs after infection.TC pigs had higher expression of MHC-I molecular,TNF-?,B cell receptor,Ig A and Ig G in the ILN control group.TC pigs also had higher expression of MHC-II molecular,T cell receptor,C3 complement,and a lot of genes related to the metabolism pathway.The cell metabolism,phagosome and NF-?B signaling all were activated in TC pigs.However,LW pigs had more down-regulated genes,TNF-?,PI3K-Akt,T cell receptor and NF-?B signaling were suppressed in LW pigs.These results suggest that the cells in ILN of TC pigs have stronger cell metabolism and higher expression of cell receptors;this may be good for TC pigs to recognize the antigen and active the downstream pathways.The suppression of MHC molecular in LW pigs has led to the failure of the activation of the cell surface receptors,resulting in PI3K-Akt,NF-?B and MAPK signaling could not operate effectively.Activation of apoptosis proteins induced by TNF-? may lead to the suppression of immune response in LW pigs.The DEGs in spleen had similar enrichment with ILN.In addition,the DEGs in the spleen of TC pigs had higher expression,which may help them to recognize the antigen quickly and active the antiviral genes.3.The proteome analysis of ILN of TC and LW pigs artificially infected with PRRSVTo validate the reliability of the transcriptome analysis of the ILN,the proteome profiling of ILN was conducted.Totally,426 differentially expressed proteins(DEPs)between the control groups,332 DEPs between the infected groups,865 DEPs in TC pigs after infection,1103 DEPs in LW pigs after infection.The DEPs were enriched in cell metabolism,phagosome,ECM-receptor and antigen presentation,which agreed with the enrichments in transcriptome analysis.TC pigs had higher expression of C3 complement,MHC family,T and B cell receptors than LW pigs before or after infection.However,a lot of proteins in ECM-receptor and G protein signaling were down-regulated in LW pigs,this may suppress PI3K-Akt signaling pathway.These pathways were consistent with the predication in transcriptome analysis.In conclusion,this study compared the differences between the TC and LW pigs infected with PRRSV through investigating clinical symptoms,blood routine index,serum cytokines,transcriptome and proteome.We found that the resistance of TC pigs to PRRSV may be associated with with their higher levels of MHC molecular,T and B cell receptors,and the stronger cell metabolism.However,suppression of ECM-receptor,PI3K-Akt,NF-?B and G protein signaling was found in LW pigs after infection,which may result in the immune suppression of them.The early maturation and high level of testosterone in the testis of TC pigs may also contribute to their resistant to PRRSV infection.In addition,this study also screened many DEGs among the main target cells,hosts‘ reproductive organ and peripheral immune organ between the two pig breeds.This study may set up a foundation for further studies investigating the PRRSV pathogenesis mechanism and providing a basis for the excavation of excellent genetic resources.