| Porcine cirovirus type2(PCV2) is the main pathogen which lead to porcine circovirus diseases(PCVD), has a worldwide distribution and is considered as one of the most important emerging viral pathogens. In recent years, more and more reports certificated that PCV2 has widely infection at home and abroad. The aim of our study was to investigate the epidemic status of PCVD in Shanghai and we have examined the epidemiology of pathogens associated with PCVD. We thoroughly investigate the genetic diversity of PCV2 strains. PCV2 is widely spreaded in the world and maintain high infection rate in herd. The serious phenomenon of PCV2 infection with other pathogens. In this study, we using the Phosphoproteomic methods to systematically investigate the global phosphorylation events in PCV2-infected pulmonary alveolar macrophages in vitro. This work effectively Provides use Phosphoproteomic-related information to facilitate further investigation of the underlying mechanism of PCV2 infection and Pathogenesis. The concrete research content is as follows: 1. the molecular epidemiological study of PCV2in 2014 to 2015, tissue samples were collected from Shanghai and surrounding areas of PCV2 testing, for PCR reaction, For the study of ORF2 genes of 10 PCV2 strains isolated from positive clinical PCV2 samples, sequencing and molecular genetic evolution analysis. The results of this research find that PCV2 d was the dominant type in sick herds in Shanghai area, existed PCV2a、PCV2b and PCV2 e in the same pig farms simultaneously.Among PCV2 with a different version of the genotypes existed the genetic sequences recombination or genetic drift in pigs. By comparing the ORF2 of PCV2 new isolates in this study, our analysis revealed the pairwise similarities of the 10 isolates ranged between 90.3% and 99% in each other. The similarity of this 10 strains with the the complete ORF2 genomes of From other regions china isolates shared were 90.3–99.3%. compared with isolates from other countries, similarities shared 90.7-99.7%. It was shown that the degree of variation of PCV2 between domestic and abroad is moderate. sequence variation of PCV2 reveal no stark regional differences on a global scale. 2. Phosphoproteome Analysis of PorcineAlveolarMaeroPhage Infection With PCV2In this experiment,The copies of PCV2 in supernatantand cells and the mRNA expression of different cytokines(IL-6、IL-8、IL-10、IL-1β) were detected by real-time PCR and IFA. On this basis, PAMs in the early stage of infection(24 h pi) and late stage of infection(60 h pi) were selected for phosphoproteomic analysis by using ITRAQ.In the study, we identified 925 unique phosphosites, of which 692 proteins and 691 proteins was significantly altered at 24 and 60 h pi, respectively. The phosphoproteomics data were analyzed to identify defined canonical pathways and functional networks. These changes in protein phosphorylation to explore PCV2 immunosuppression and pathogenic mechanism provides a number of useful information, which were regulated by several protein kinases during virus infection. We found that PCV2 in the infected host cells, inhibiting apoptosis, promoting autophagy, may be due to the Hippo and the activation of Notch signaling pathways and phosphoinositide signal, change some of the protein kinase. Interacting network analysis indicated that altered phosphoproteins were involved in Assembly and component, signal transduction, protein synthesis, nucleic acid metabolism, metabolism, cell activity, etc. These pathways and functional networks analysis could help us elucidate the pathogenic mechanisms of PCV2 infection. The exact role of these differential phosphoproteins in uninfected PAMs andPCV2-infected PAMs still needs to be determined further. |
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