| Porcine parvovirus(PPV)is one of the main pathogens that cause reproductive disorders in pigs,causing abortion,stillbirth,mummification,deformity and weak fetus in sows.At present,the prevalence of PPV is extremely widespread and can be detected in various countries around the world.Due to the strong resistance of PPV to the external environment,it is difficult to remove the virus completely after the pig farm is infected with PPV.The mixed infection of PPV and other viruses also causes more serious impact.PPV infection has no obvious seasonality and a high infection rate in China,which has caused huge economic losses to China and the world's pig industry.Therefore,the monitoring and early diagnosis of the virus is particularly important.It has been listed as class ii animal epidemics by the ministry of agriculture of China.There has been relatively little continuous detection and research on the virus and the understanding of the mechanism of PPV infected cells is still incomplete since the discovery of PPV.Hence,in order to detect PPV infection in pig population quickly,we have established a fluorescent quantitative detection method that can detect PPV quickly and accurately to provides a technical basis for monitoring the prevalence of PPV.For further understand the interaction between PPV and cells,we used proteomics technology to analyze the differential protein of PK-15 cells infected with PPV at different times,providing data support for the exploration of PPV infection and pathogenesis.1.Because of the advantages of real-time fluorescent quantitative PCR,such as fast,accurate and specific,it is widely used in the detection of pathogens.In order to monitor the infection status of PPV in pigs,this experiment established a fluorescence quantification method for the rapid detection of PPV infection in pigs by optimizing the amplification conditions of real-time fluorescence quantification.The results showed that the method had high specificity,sensitivity and repeatability.Testing of clinical samples from aborted piglets and sows revealed the presence of PPV in breeding failure pigs in farms in Henan province.The positive rate of fluorescent quantitative PCR was 25%,and the positive rate of ordinary PCR was 14.6%,which proved that the sensitivity of the method was higher than that of ordinary PCR.2.In this study,Tandem Mass Tag(TMT)and Liquid Chromatography-tandem mass spectrometry(LC-MS/MS)were used to quantify the differentially expressed proteins in PK-15 cells infected by PPV for 6h and 36h respectively.Firstly,we determined the dynamic growth curve of PPV and observed the cytopathic changes of PK-15 cells at different time points after PPV infection.We selected the time points of PPV infection for 6 hours and the highest copy number of virus and obvious cytopathic changes for proteomic identification.The results showed that the copy number of virus particles reached the maximum at 36h after PPV infection,and the pathological changes were obvious.Therefore,the samples infected with PPV for 6 h and 36 h were selected for subsequent tests.According to proteomic analysis,the length of identified peptide segments was mainly less than 25aa,20935 in total,accounting for 99.22%of the total identified peptide segments.The identified peptide coverage was mainly center at below 10%,corresponding to 2397 proteins,which was 50.26%of the total protein screened,,and the identified maximum peptide coverage was 87.25%.The molecular weight of the identified proteins was mainly below 80kDa,3643 in total,accounting for 76.39%of the total identified proteins.The differentially expressed protein was screened by the standard of multiple change more than 1.5 times(up-regulation more than 1.5 times or down-regulation less than 0.67)and P value less than 0.05.According to statistics,32 differential proteins were screened in the 6h group of PPV infection,among which 21 proteins were significantly up-regulated and 11 proteins were significantly down-regulated.A total of 345 differential proteins were screened at 36 hours after PPV infection,of which 146 were significantly up-regulated at 36 hours after infection and 199 were significantly down-regulated at 36 hours after infection.3.In order to investigate the biological processes and signaling pathways involved in the different proteins screened after PPV infection of pk-15 cells,GO annotation and KEGG signaling pathway enrichment were performed on the different proteins.The results showed that the biological process of differential protein in PK-15 cells infected with PPV for 6 hours mainly included single-organism process,metabolic process,localization,cellular component organization or biogenesis,immune system process,locomotion,biological adhesion.The cell components of differential proteins were mainly concentrated in cells,cell parts,organelles.The molecular functions of different proteins are mainly focused on binding,catalytic activity and transport activities,etc..The signal pathways of significant enrichment of differential proteins mainly include mineral absorption,Toll-like receptor signaling pathway,TNF signaling pathway,viral carcinogenesis,etc.,involving cell growth and metabolism,protein transport,immune response,cytoskeleton change.The biological process of PPV infected PK-15 cells in 36h group was mainly concentrated in cellular process,metabolic process,cellular component organization or biogenesis,localization,immune system process,locomotion,cell killing and detoxification.The cell components of differential proteins were mainly concentrated in cells,cell parts and organelles,The molecular function of differential proteins was mainly concentrated in binding,catalytic activity,transport activity,molecular function regulator,etc..Significantly enriched signaling pathways which mainly include gap junction,oxidative phosphorylation,sphingolipid signaling pathway,spliceosome,phagocyte,viral carcinogenesis,tight junction,B cell receptor signal pathway,etc.,involving intercellular information transmission,protein modification,RNA modification,immune process,etc..4.In order to compare the differential proteins at different time points after PPV infection and the main physiological processes involved,we analyzed the proteins identified at 6 h and 36 h after PPV infection,with multiple change more than 1.5 times(up-regulation more than 1.5 times or down-regulation less than 0.67)and P value less than 0.05.The results showed that there were 187 differentially expressed proteins,120 up-regulated proteins and 67 down-regulated proteins with PPV for 36 h and 6 h.The biological processes in which differentially expressed proteins are involved mainly include single-organism process,metabolic process,cellular component organization or biogenesis,localization,developmental process,immune system process,and cell killing.The molecular functions of different proteins mainly include binding and catalytic activity,etc..The pathways of significant enrichment mainly include sterid biosynthesis,pentose and glucuronate interconversions,adherens junction,endocytosis,spliceosome,etc.,indicating that PPV has significant differences in biosynthesis,cytoskeleton,RNA processing etc.,compared with 6h and 36h after infection. |
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