Proteomic Analysis Of PK-15 Cells Infected With Pseudorabies Virus Variant And Development Of A High-temperature Passaging Attenuated Pseudorabies Vaccine

Pseudorabies?PR?,a serious infectious disease of swine,with the characteristics of high mortality in young piglets and reproductive failure in sow,results in tremendous economic losse in swine industry.The causative agent of PR is pseudorabies virus?PRV?.The process of PRV infecting host cells is very complex and the mechanisms of PRV pathogenicity have not been fully understood,which made it difficult to find new targets for the disease diagnosis and prevention.At present,vaccination is the most effective way to control PRV infection.However,PR outbreaks have occurred in Bartha-K61-vaccinated pigs on many Chinese farms and caused huge economic losses since 2011.The PRV variant was shown to possess increased virulence in pigs.Bartha-K61 vaccine did not provide full protection against the PRV variant.In this study,proteomics method was used to analyze the changes of cellular proteins expression between the PRV JS-2012 infected and uninfected PK-15 cells.The data were analyzed and verified,and the results were given below.In addtion,PRV JS-2012 was continuously passaged in Vero cells at 40°C,and an attenuated strain JS-2012-F120 was obtained.It was tested for the efficacy in piglets.The main works and results of this study were as follows:1.PK-15 cells were harvested at 2 h,4 h and 7 hpi with PRV variant strain JS-2012 and an iTRAQ-based comparative quantitative proteomic study was performed.A total of 71,198and 122 differentially expressed proteins were identified,respectively.For biological process annotation,proteins in 2 h group were mainly involved in intracellular protein transport,transcription,signal transduction and positive regulation of transcription.Proteins in 4 h group were mainly involved in regulation of transcription,signal transduction,apoptotic process and immune response.Proteins in 7 h group were mainly involved in immune response,actin filament organization,multicellular organism development and vesicle-mediated transport.An STRING analysis of the immune related proteins which were screened from all differentially expressed proteins showed that the protein SRC was the center of the network and had a potential interaction with other proteins.In this study,the proteomic analysis to compare whole cellular protein alterations induced by PRV variant strain infection,might contribute to understand the pathogenesis of PRV and anti-viral strategy development.2.PRV JS-2012 was continuously passaged on Vero cells for 120 times.An gE-deleted attenuated strain,JS-2012-F120 was derived from the 120th passage virus by rounds of plaque cloning.Sequence analysis showed that JS-2012-F120 contained a 2307-bp deletion covering nucleotide 487 of gE gene to 531 of US2 gene,and IFA confirmed that the gE proteins of JS-2012-F120 were not expressed.JS-2012-F120 was showed notably different cytopathic effects and plaque morphology from JS-2012.JS-2012-F120 prone to forming spindle shaped plaques,while the plaque morphology of JS-2012 was nearly round and the plaque sizes of JS-2012-F120 were larger than those of JS-2012.JS-2012-F120 grew faster than JS-2012,and the peak titer of JS-2012-F120(10^9.25 TCID₅₀/ml)was significantly higher than that of JS-2012(10^8.375 TCID₅₀/ml).3.To evaluate the virulence of the different passaged viruses in piglets,10⁶ TCID₅₀ of JS-2012-P5H,JS-2012-P50H,JS-2012-P91H or JS-2012-P120H?JS-2012-F120?was used to inoculate piglets.After inoculation with JS-2012-P5H,all piglets developed typical PR symptoms and eventually died at 4-5 dpi.The virulence of JS-2012-P50H partly declined.Although all piglets displayed PR symptoms,the severity was markedly reduced and three of five piglets survived throughout the experiment.All five piglets infected with JS-2012-P91H survived throughout the experiment.One piglet showed no clinical symptoms while the other four piglets had a fever.All piglets survived throughout the experiment and no clinical symptoms were observed in piglets infected with JS-2012-P120H.The surviving piglets?killed at 21 dpi?and dead pigs were necropsied.All dead piglets and the piglets undergoing a fever showed blood spots and necrosis in the lungs,and had spleen infarcts with blood spots and hemorrhagic lymph nodes.There were no visible pathological lesions in the organs of the piglets infected with JS-2012-P120H and the piglet without fever infected with JS-2012-P91H.4.To evaluate the efficacy of JS-2012-F120 immunization against challenge with the classical or emerging PRV strain,2-week-old piglets were inoculated with 10⁵ TCID₅₀ of JS-2012-F120 or DMEM.Four weeks post-vaccination,immunized and unimmunized groups were both challenged with the classical virulent PRV strain SC or PRV variant JS-2012.All the immunized piglets were survived after challenge with JS-2012 or SC and no clinical PR symptoms were observed.The gE antibody remained negative in immunized piglets.However,all piglets in the unimmunized displayed typical PR symptoms,and five piglets challenged with JS-2012 died and four piglets challenged with SC died eventually.Thus,vaccination with JS-2012-F120 provides full protection for piglets against challenge with classical and emerging virulent PRVs.JS-2012-F120 appears to be a promising marker vaccine to control PRV variant circulating in Chinese pig farms.