Studies On Molecular Epidimiology Of PCV2and The Interaction Of JAK-STAT Signal Pathways With Infection Of PCV2

Porcine circovirus (PCV) is a nonenveloped, icosahedral virus containing a circularsingle-stranded DNA genome, which is one of the minimum DNA animal viruses. Porcinecircovirus is divided into two serotypes: PCV1and PCV2, PCV1is not pathogenic whilePCV2is pathogenic. The spread of PCV2causing huge economic losses to the pig industry.Ithas seriously hampered the development of the pig industry.More than a dozen provinces reported porcine circovirus virus type2(PCV2) infectionssince porcine circovirus virus type (PCV) antigen has been detected by Lang Hongwu firstlyin2000. It spread gradually and seriously hampered the healthy development of pig industryof China. Currently, the pathogenesis of PCV2is still at the exploratory stage.The study ofcellular signal transduction caused by PCV2infection will help to elucidate the pathogenesisof PCV2.There are no reports about the way JAK-STAT signaling pathway in PK-15cellsinfected with PCV2curbing viral protein expression and virus proliferation.To understand thegenetic variation of PCV2epidemic strains in Shandong Province the molecular prevalence ofPCV2in Shandong Province from2010to2012was investigated. The whole genomesequence of PCV2isolates of Shandong have been compared to the submitted sequence of theGenBank with the whole genome sequence.PK15cells were infected with PCV2isolatedfrom diseased or serum which antigen test is positive. The relationship between JAK-STATsignaling pathway and PCV2infection was studied by PCV2-specific fluorescencequantitative PCR and PK-15cells α-IFN real-time quantitative RT-PCR.There are four parts of this study.1. Epidemiological investigation proliferation properties of porcine circovirus virus type2in Shandong provinceIn order to understand PCV2infection situation of swinery in Shandong province inrecent years, we collected lung, lymph nodes, spleen and other organs of the pathologicalchanges, anticoagulant, serum from2010to2012December from300big farms in Shandongprovince, and detected the antigen and antibody of PCV2using PCR and ELISA method. Theresult of survey showed that the antibody positive rate in2010,2011,2012was72.95%,61.85%,59.89%, while antigen positive rate was34.66%,29.59%,21.38%, respectively. Inaddition, the PCV2antibody levels of serum in pig farms which did not immune PCV2 vaccine were as high as88.72%. This study was aimed at analyzing systematically PCV2infection and popular rule in pigs in our province, which provide reference for prevention andcontrol of PCV2. PK15cells were infected by tissues or serum which was positive in PCV2antigen detection.The virus isolates were obtained, and the titer of virus generation in vitro was assayed.The results of tests showed that two isolates were obtained from tissues or serum. In theindirect immunofluorescent assay (IFA) test, the nucleus sent dense green fluorescents andcytoplasm appeared sparse green fluorescent, while there were no fluorescents in control cells.The growth curve of PCV2isolated strain (SD-JN) was detected. The result showed that theTCID50of PCV2achieved the peak when PK15growth to72hours, and then began todecline.The result of animal regression test showed that Kunming mouse infected PCV2andcontrol mouse did not appear obvious clinic symptoms, but the result of PCR was positive intest group. The obtaining of PCV2isolates laid a solid foundation for the research of theproliferation characteristics of viruses in vitro, to clarifying the mechanism of interactionbetween PCV2and PK15cells, to develop further high quality vaccine.2.Spatial and temporal distribution and genetic evolution of porcine circovirus type2andthe genome sequence of PCV2b virus isolates from Shandong provinceThe study of amplification cloned whole genome sequence of PCV2from sick pig in thetissue, blood, and26strains of isolates in Shandong from our laboratory were sequenced andcompared with the sequence of PCV2isolates submitted in the GenBank, the results showedthat26strains of Shandong PCV2isolates nucleotide homology of69.9%-69.9%, amongisolated strains of DQ478947in2006and separate strain of HM142894in2010of homologylowest.The whole genome of90strains PCV2from the isolated gene sequences downloadedfrom Shandong Province26PCV2gene sequences and in GenBank (26strains of otherprovinces in mainland China, Taiwan7strains and30strains in foreign) were analyzed towhole sequence of nucleotides and ORF2amino acids encoded by gene phylogenetic tree.The results show that53PCV2of the separation from mainland China, PCV2b subtypes of45strains, subtype PCV2a of8strains, full description PCV2b subtypes. At present, the domesticmainland major PCV2major pandemic strain. From looking at the year of separation, since1998,the ring virus epidemic strain went from PCV2a as the main epidemic strains,thecoexistence of PCV2a and PCV2b subtypes, with PCV2b as the main evolution of theepidemic strains.On the genetic tree, currently the isolation and identification of PCV2bsubtype of HIV strains, with Qinling huaihe River as the boundary, in accordance with the region divided into southern China and Northern China two large clusters.From ORF2evolution tree analysis, isolates from Shandong24PCV2ORF2gene concentrated on the twobranches of both ends of the evolutionary tree, which9Article Shandong separation strains ofORF2gene is located in evolution tree of the upper part of Zone A branch and France, andHungary separation strains genetic distance more near,15article Shandong separation strainsof ORF2gene is located in evolution tree of the lowe part of zone B branch, and UnitedKingdom separation strains genetic distance more near. The purpose of this test is tounderstand the genetic variation of PCV2epidemic strains of Shandong Province, to provide atheoretical basis for the diagnosis of porcine circovirus disease and vaccine research anddevelopment.3.The interaction of JAK-STAT signaling pathways with infection of PCV2Specificity primers were designed according the gene sequences of β-actin、Mx1、OASfrom pig in the GenBank for β-actin、Mx1、OAS from PK-15cell and a real-time RT-PCRmethod was established in our lab. The best action time and the optimal concentration aboutα-IFN in PK-cell was defined that were750U/ml and12hours.The best time to gather thePCV2from PK-cell is72hours post infection. The best action time and the optimalconcentration about AG490(special inhibitor to JAK-STAT pathway) in PK-cell wereconfirmed by real-time RT-PCR method of β-actin、Mx1、OAS and PCV2. The results showedthat the optimal condition is10μM and12hours. The expression level of Mx1、OAS geneswere tested in different time. The results are that the genetic expressions of Mx1、OASincreased after PCV2infection in PK-cell than the control PK-cell. But it is lower than thegenetic expressions of Mx1、OAS in PK-cell treated with the α-IFN. We can confirm that theinfection of PK-cell can activate the JAK-STAT singaling Pathway. There are high level ofPCV2in the PK-cell in which the JAK-STAT pathway was inhibitted completely by AG490than the PK-cell as control. And there are low level of PCV2in the PK-cell in which theJAK-STAT pathway was activated completely by α-IFN than the PK-cell as control.Then,wecan determine that JAK-STAT pathway activation can inhibit the proliferation of PCV2.This conclusion can help us to study the biological mechanism of PCV2infection.