The Ers-mediated Autophagy Induced By CSFV And The Effect Of ERS On Immune Effect Of A CSF E2 Subunit Vaccine Candidate

Classical swine fever（CSF）,caused by classical swine fever virus（CSFV）,is an acutely fatal disease of pigs or wild boars.CSFV infection causes high fever,hemorrhagic necrotizing inflammation in multiple organs of pigs with a high mortality rate,which poses a great threat to the pig industry.Although the prevention and control of CSF has made great progress,the pathogenic mechanism of CSFV remains unclarified.Endoplasmic reticulum stress（ERS）and autophagy can be employed as important regulatory mechanisms of protein homeostasis and intracellular metabolism in eukaryotic cells.Many mammalian viruses can affect the innate immunity and apoptosis of host cells by regulating ERS and autophagy,to facilitate viral infection.However,the relationship between ERS,autophagy and virus replication during in vivo and in vitro infection of CSFV has not been fully elucidated.Based on the combination of both in vivo and in vitro experiments,here we reveal the relationship between CSFV-induced ERS,autophagy,viral replication,and innate immunity,which will provide a scientific reference for further research on the pathogenesis of CSFV.Study on the relationship between tissue lesions,viral loads,and ERS-autophagy in CSFV-infected pigs.Firstly,we established a pig model for CSFV infection by monitoring the body temperature,clinical symptoms,pathological changes,and viral infections of the experimental pigs.Histopathological studies showed that CSFV infection caused various degrees of hemorrhagic and necrotic inflammation in different organs,among which the tonsil,lymph nodes,spleen,thymus,kidney,lung,and intestine possessed more serious histopathological injuries than heart,liver,and brain.Results showed that loads of CSFV in tonsils,lymph nodes,spleen,thymus,kidney,lung,and intestine were significantly higher than those in heart,liver,and brain,suggesting that the degree of tissue lesions was to some extent positively correlated with CSFV replication in vivo.Western blot analyses showed that CSFV infection not only induced ERS and subsequently activated three unfolded protein responses（UPR）pathways including protein kinase R-like ER kinase（PERK）,inositol requiring enzyme 1（IRE1）and activating transcription factor-6（ATF-6）pathways,but also triggered complete autophagy in tonsils,lymph nodes,spleen,thymus,kidney,lung,and intestine,which exhibited severer pathological injuries and higher viral loads.However,only the IRE1 pathway and no autophagy were activated in heart,liver,and brain with slighter pathologies and lower viral loads.Collectively,we inferred from the above results that differential tissue lesions and viral loads appeared in the CSFV-infected pigs may be related to ERS and autophagy.Study on the relationship between ERS and autophagy during CSFV infection.We further carried out in vitro experiments to verify this inference.First,we performed q RT-PCR and Western blot assays to detect the expression of ERS and autophagy indicators in cultured porcine kidney cell lines（PK-15）and macrophage cell lines（3D4/2）infected with CSFV.We found that CSFV infection activates the PERK and IRE1 pathways and complete autophagy in the cultured PK-15 and 3D4/2 cells.Further study showed that pretreatment with ERS activator（Thapsigargin/TG）remarkably enhanced while ERS inhibitors（TUDCA and 4PBA）significantly downregulated autophagic activities in the CSFV-infected cells in a dose-dependent manner,suggesting that CSFV-induced autophagy could be mediated by ERS.To detect the effect of ERS modulators on autophagic flux in the CSFV-infected cells,PK-15 cells were pretreated with TG,TUDCA or 4PBA,and then transfected with an autophagy dual fluorescent reporter plasmid（m RFP-GFP-LC3）prior to CSFV infection.The autophagosomes and autophagolysosomes in the cells were observed under a confocal microscope.Results showed that TG significantly increased the numbers of autophagosomes and autophagolysosomes in the CSFV-infected cells,suggesting elevated autophagic flux;both TUDCA and 4PBA significantly reduced the numbers of autophagosomes and autophagolysosomes in the CSFV-infected cells,suggesting decreased autophagic flux.The above results indicated that CSFV infection induces ERS-mediated complete autophagy.In the meantime,we found that TG treatment increased while TUDCA or 4PBA decreased the transcriptional levels of CSFV NS5B genes,expression of CSFV N^pro proteins,and viral titers in CSFV-infected PK-15 and 3D4/2 cells,implying that viral replication in the cultured cells was regulated by CSFV-induced ERS.We also found that autophagy activator（Rapamycin/RAPA）significantly increased while autophagy inhibitor（3-Methyladenine/3-MA）remarkably reduced the copy numbers of CSFV NS5B genes and virus titers in the CSFV-infected cells,indicating that CSFV-induced autophagy was beneficial for viral replication.To further confirm the relationship between ERS-mediated autophagy and CSFV replication,PK-15 and 3D4/2 cells were pretreated with TG or TUDCA,followed by treatment with RAPA or 3-MA prior to CSFV infection.q RT-PCR and TCID₅₀ tests were used to detect copy numbers of CSFV NS5B genes and virus titers in the CSFV-infected cells.Results showed that RAPA promoted CSFV replication in the infected cells,and treatment with TG significantly enhanced while TUDCA remarkably suppressed RAPA-promoted CSFV replication.We aslo showed that 3-MA inhibited CSFV replication in the infected cells,however,TUDCA further decreased and TG efficiently reversed 3-MA-reduced CSFV replication in the infected cells.Collectively,our data demonstrated that CSFV induces ER stress-mediated autophagy to sustain its replication in vivo and in vitro.To study the mechanism of ERS-mediated autophagy during CSFV infection,cells were treated with PERK agonist（CCT020312/CCT）,PERK inhibitor（GSK2606414/GSK）,e IF2αdephosphorylation inhibitor（Salubrinal/SAL）,IRE1 inhibitor（4μ8c）or GRP78 inducer（BIX）,or PERK/IRE1/GRP78 gene-targeting interfering RNA,followed by CSFV infection.Transcriptional and translational levels of ERS-,autophagy-,and CSFV-genes,as well as virus titers were detected by q RT-PCR,Western blot,and TCID₅₀assays,respectively.Results showed that CCT,SAL,and BIX treatments promoted autophagy and viral replication in the CSFV-infected cells.Contrastively,GSK and 4μ8c treatments,as well as the PERK/IRE1/GRP78 gene silencing significantly inhibited autophagy and viral replication in the CSFV-infected cells.These results suggested that CSFV infection induces PERK-and IRE1-dependent autophagy to promote viral replication.We further found that CCT,SAL,and BIX treatments increased while GSK and 4μ8c treatments,as well as the PERK/IRE1/GRP78 gene silencing reduced autophagic flux in the CSFV-infected PK-15cells.These results suggested that CSFV infection induces PERK-and IRE1-dependent complete autophagy,facilitating CSFV replication in vitro.In addition,we also showed that CCT,SAL,and BIX treatments increased,while GSK,4μ8c treatment,and PERK,IRE1 and GRP78 gene silencing obviously decreased the transcription of IFN-γand TNF-αgenes in the infected cells,indicating that activation of the PERK and IRE1 pathways induced by CSFV infection not only promoted autophagy and viral replication,but also was involved in immune regulation in the CSFV-infected cells.Study on the effect of ERS regulators on the immune effect of CSF E2 subunit vaccine candidate.Considering the potential immunomodulatory properties of ERS regulators,we explored the effects of combination of ERS modulators（TG,TUDCA and4PBA）and a CSF E2 subunit vaccine candidate on the immune effect in experimental New Zealand rabbits.Indirect ELISA was used to detect the titers of CSFV E2-specific antibodies in sera from the immunized rabbits.Proliferation and the transcriptional levels of Th1-（IFN-γ,TNF-α,IL-2）and Th2-type cytokines（IL-4,IL-6,IL-10）of peripheral blood mononuclear cells（PBMC）were respectively detected by CCK8 and q RT-PCR assays.Furthermore,typical fever reaction and tissue viral loads in the immunized rabbits were also detected.On the one hand,results showed that vaccination of the CSF E2 vaccine candidate stimulated rabbits to produce continuous and high-titer E2-specific antibodies,and at the meantime enhanced the proliferative activity and transcription of Th1-（IFN-γ,TNF-α,IL-2）and Th2-type cytokines（IL-4,IL-6,IL-10）in PBMC,suggesting that vaccination of E2 subunit vaccine evoked effective humoral and cellular immune responses.Moreover,immunization-challenge experiment showed that a typical fever reaction appeared in the blank rabbits after being challenged with CSFV-C strain,and copy numbers of CSFV-NS5B genes in spleen,lymph node and PBMC from the blank rabbits were relatively higher.In contrast,no typical fever was obtained,and no viral loads was detectable in spleen,lymph node,kidney,and PBMC from the rabbits immunized with the CSF E2 subunit candidate or with the CSFV-C strain.Results suggested that immunization of the CSF E2 subunit vaccine candidate provides complete protection against challenge with CSFV-C strain.On the other hand,ERS modulators effectively regulates the cellular immunity in the E2-vaccinated rabbits,although they seemingly have no effect on the E2-evoked humoral immunity.In addition,combination of ERS modulators and E2 vaccine also provided complete protection against the challenge with CSFV-C strain.Collectively,the above results confirmed the safety and effectiveness of the CSF E2 subunit vaccine candidate and the potential of ERS regulators as immunomodulatory drugs for controlling CSF.In summary,through combination of in vivo and in vitro studies,we reveal that CSFV infection induces ERS,and then activates the PERK-and IRE1-mediated complete autophagy to promote virus replication;meanwhile,the PERK and the IRE1 pathway may be involved in regulating cellular immune responses.Our findings will lay foundations for elucidating the pathogenic mechanism of CSFV infection and developing new strategies to effectively prevent and control CSF.In addition,ERS modulators can effectively regulate the cellular immune responses stimulated by immunization with a CSF E2 subunit vaccine candidate,providing a reference for screening immunomodulatory drugs for controlling CSF.