Metabonomics Study Of Pseudorabies Virus Infected Porcine Alveolar Macrophage And Construction Of A Novel Vaccine By Targeting Dendritic Cells

The main clinical symptom of porcine pseudorabies(PR)is characterized with high fever,cough,reproductive disorders and acute encephalomyelitis.Owing to the national PR eradication program and vaccination for pseudorabies virus(PRV)in large-scale pig farms,PR was only sporadically reported in China before 2011,and most reported cases were latent infections.In November 2011,the first case of emerging PRV infection was reported in Tianjin.Since then,emerging PRV strains have caused unprecedented outbreaks in pig farms in more than 20 provinces,cities and autonomous regions.Bartha strain-and classical strain-based vaccines don’t provide effective protection against emerging PRV infection,and many PRV infection cases have been reported in vaccinated pigs.With the improvement of viral sequencing technology,the differences in gene sequences among PRV vaccine strains,classical strains and emerging strains have been clarified,but the pathogenic mechanism which cause the differences among different PRV strains is still unknown.This study aimed to identify the potential pathogenic differences and metabolic characteristics of different PRV strains by metabonomics analysis.The results of this analysis were used to develop a novel vaccine with high efficiency and reliability against emerging PRV infection.Here,the main results are listed as follows:1.PRV strains alter cellular lipid metabolism in infected macrophage.In February 2020,a total of 31 PRV whole genome sequences were available in the NCBI database.Based on these sequences,a PRV whole genome evolutionary tree was constructed.The results showed that 12 foreign isolates,including Bartha strain,were located on the same evolutionary branch.Nineteen strains of PRV isolated from China were located on a separate evolutionary branch.The Chinese PRV strain evolutionary branch was further divided into two subbranches.Four out of 19 PRV strains,including the EA strain,belong to the same sub-branch,and they are considered as classical strains.However,the strains isolated after 2011 belong to the other Chinese PRV strain sub-branch,and they are considered as emerging strains.Based on this evolutionary analysis,a vaccine strain(Bartha),a classical strain(EA)and an emerging strain(HNX)were used to research the metabolic characteristics of different PRV strains.The growth characteristics of Bartha strain,EA strain and HNX strain in iPAM were calculated.The metabolites produced by PRV vaccine strain(Bartha)-,classical strain(EA)-and emerging strain(HNX)-infected iPAMs were analyzed by liquid chromatography-mass spectrometry(LC-MS)and bioinformatics techniques.A total of 3864 metabolites were identified.Compared with the uninfected cells,levels of 246 metabolites were altered in the Bartha strain-infected cells,225 were altered in the EA strain-infected group,and 272were altered in the HNX strain-infected group.In Bartha strain-,EA strain-and HNX strain-infected iPAM cells,changes in the levels of lipids accounted for over 50%of the altered metabolism.Glycerolipids,the main lipids in the cell membrane,changed the most,and accounted for over 27%of all metabolic alternations.PRV,as an enveloped virus,is strictly dependent on the host’s cellular membrane systems for its replication.The abundance of phosphatidylserine and phosphatidylethanolamine,which are the main components of cellular rafts,significantly decreased in all the infected iPAM cells.However,the HNX strain consumed more phosphatidylserine and phosphatidylethanolamine than the Bartha and EA strains.We also found that different PRV strains had a priority for the utilization and consumption of some specific types of molecules which have the same carbon chain.The abundance of lipids with roles in innate immunity and inflammation,such as sphingomyelin,ceramide,galactosylceramide and glucosylceramide,also changed during PRV infection.The HNX strain induced metabolic alternations may regulate the cellular biological characteristics and provide advantageous conditions for virus replication.2.Long chain ceramide suppress PRV replication by inducing M1 polarization of iPAM cells.The results of the metabonomic analysis revealed metabolic alternations common to all three PRV strain infections.However,we also found strain-specific metabolic alterations,such as a decrease in some long-chain ceramides in HNX strain-infected iPAM cells.Despite being an important bioactive metabolite,the influence of long-chain ceramide on PRV replication and antiviral immune responses are still unknown.To investigate the potential influence of long-chain ceramides on PRV infection and polarization of porcine alveolar macrophage,iPAM cells were treated with C14-ceramide and classical M1polarization marker gene transcription(IL-1β,TNF-α,IL-6 and IL-12)was examined using RT-qPCR.The influence of Toll-like receptor 4(TLR4)on macrophage polarization was similarly explored using the TLR4 inhibitor(Resatorvid)and silencing of myeloid differentiation factor(MyD88)transcription using siRNA.The results showed that C14-ceramide induced M1 polarization of macrophages via TLR4-MyD88 pathway.We found that stimulation with C14-ceramide induced M1 polarization of iPAM cells and significantly inhibited the replication of Bartha,EA and HNX PRVstrains.We also found that C14-ceramide could induce the expression of Fms-like tyrosine kinase 3 ligand(Flt3L)in iPAM cells.3.The potential of Flt3L as a molecular adjuvant in PRV vaccine development.Previous reports showed that Flt3L,which we found was induced by C14-ceramide stimulation,is a regulatory molecular in antiviral innate immunity.It also shows the potential as a molecular adjuvant in vaccine development.We therefore explored the potential of Flt3L as a molecular adjuvant in PRV attenuated vaccine development.CRISPR/Cas9 and Cre/lox systems were used to construct an HNX-TK~-/g E~--Flt3L recombinant virus.RT-qPCR,Western blot and immunofluorescence staining were used to calculate the m RNA transcription and protein expression of Flt3L in HNX-TK~-/g E~--Flt3L infected cells.The growth characteristics of the recombinant virus,HNX-TK~-/g E~--Flt3L,were determined in vitro by one-step growth curve assay in iPAM cells,which showed no significant difference between the HNX-TK~-/g E~--Flt3L strain and its parent strain.We also found that,compared with traditional TK and g E attenuated strains,the immunogenicity of HNX-TK~-/g E~--Flt3L was significantly increased.In HNX-TK~-/g E~--Flt3L infected mice,the levels of neutralizing antibody,PRV-g B antibody and interferon were significantly increased.Further studies showed that the HNX-TK~-/g E~--Flt3L could activate dendritic cells in vitro and in vivo.It also showed better protection against HNX challenge infection in mice.In summary,our results showed that Flt3L is an ideal molecular adjuvant in PRV vaccine development.HNX-TK~-/g E~--Flt3L showed the potential to be a promising and efficient PRV vaccine against emerging PRV strains.