Identification Of Putative ORF4Within Porcine Circovirus Type2and Gene Expression Profile Of Host Immune Organ After PCV2Infection

Porcine circovirus type2(PCV2) is the primary causative agent of porcine circovirus-associated diseases (PCVAD) in pigs.To date, three proteins encoded by ORF1,ORF2and ORF3have been identified.ORF1encoded a replication-related proteins Rep, ORF2encoded an capsid protein Cap, and ORF3encoded an apoptosis inducer called ORF3protein.In this study, a novel transcription unit and protein (termed ORF4here) was detected during productive infection of PCV2in PK15cells Meanwhile gene expression profile of host immune tissue after PCV2infection was studied by affymetrix gene chip.What has done was try to study the roles of PCV2encoded protein in pathogenicity and the host response to PCV2infection.1. Identification of PCV2ORF4protein and transcription in PCV2infected PK15cellsIn this study, potential epitope of PCV2ORF4protein was predicted by BepiPred1.0software and peptide containing the epitope was synthesized by chemical synthesis.Peptide and eukaryotic expression plasmid pCI-ORF4was used to immune BALB/C mice,ORF4specific monoclonal antibody and polyclonal antibody were prepared.Using the above antibodies in IFA specific signal was detected in PCV2infected and pEGFP-ORF4transfected PK15cells while none signal was detected in normal PK15cells and pEGFP-ORF1,2,3transfected PK15cells,indicating that a novel protein has exist in PCV2infected cells independent of Rep, Cap and ORF3protein.Northern blot results show that, ORF4corresponding transcripts is about180bp, using the different start codon of ORF3, which overlapped in ORF3RNA and has the same transcription direction of ORF3. The above results has identified a novel protein and transcript unit in PCV2and provided a favourable backround for he follow-up study.2. Analysis of ORF4function both in vivo and in vitroTo analyze the function of PCV2-ORF4encoded protein, ORF4deficient PCV2(PCV2A) was constructed and virus was sucessfully rescued which indicating ORF4protein is not essential for PCV2replication. PCV2A and wild type PCV2(rPCV2) were used in PK15cells and mouse to study the function of ORF4protein.The results showed that, PCV2A infected cells can cause the activity of caspase-3,8,9increased which suggested ORF4protein is associated with apoptosis inhibition.With regard as serum viral load, PCV2△infected mice increased about20to100times to that of wPCV2and show more severe lymphoid tissue damage in early stage. Meanwhile PCV2△caused a more severe decreased of CD3+CD4+, CD3CD8+, CD4+CD8+T lymphocytes. The results indicated that with the absence of ORF4protein, virulence of PCV2was enhanced and ORF4protein maybe a negative regulator in PCV2pathogenicity.3. Gene expression profile of host immune organ after PCV2infectionTo analyze the response of immune organs to PCV2infection, gene chip was used to study the transcriptome in PCV2-infected pigs lymph nodes and spleen PCV2-infected mouse. The results show that66different expression transcripts were found in inguinal lymph node of PCV2infected pigs including44upregulated transcripts mainly related to macromolecular metabolism (20%), differentiation and development (18%), transport(13%) and cytoskeleton-related transcripts(7%), and22down regulated transcripts mainly related to immune/inflammatory responses (57%) and metabolism (9%). In spleens of PCV2infected mice,137different expression transcripts were found including40upregulated transcripts mainly related to metabolism (12%), differentiation and development (12%), immunization (10%),signal transduction (10%) and biological macromolecules biosynthesis (10%).73down regulated transcripts mainly related to metabolism (13%), differentiation and development (15%),immune response (12%), macromolecule biosynthesis (11%) and signal transduction (11%). Quantitative PCR was used to cofirm the microarray result,apart from down-regulated genes in mice spleen the other result of microarray and RT-PCR are basically concordant. Finally western-blot was use to detect the protein level of cofilinl and DSTN in non-phosphorylated form,results showed that the non-phosphorylated form of cofilinl and DSTN was upregulated in spleen, inguinal lymph nodes and lymphonodi mesenterici in PCV2infected pigs.The resut indicated that PCV2infection may lead actin depolymerization to host cells by up regulation of depolymerization factor.