porcine circovirus ( pcv ) , a member of the genus circovirus in the family circoviridae , is a small non - enveloped virus with a single - stranded circular dna genome of approximately 1.8  kb . to date , two distinct genotypes of pcv have been identified : pcv1 and pcv2 . pcv2 has been regarded as a major pathogen of pcv associated diseases ( pcvad ) , of which postweaning multisystemic wasting syndrome ( pmws ) is the major syndrome , having a severe economic impact on swine production wordwide , . pcv - like agent p1 , discovered in 2007 from the serum of the porcine with pmws , contains a circular dna molecule of 648 nucleotides in length , which is highly homologous to the genome of pcv2 with the exception of 16 nucleotides . our previous study confirmed that the p1 virus has been epidemic in china 's pig farms and can infect pigs displaying pmws - like signs , . the genome of p1 contains eight open reading frames ( orfs ) . in this work , a tandem dimmer of p1 genome was cloned into the bamhi site of bluescript plasmid ( invitrogen ) , which was regarded as the  parent  to construct all other p1 mutant plasmids . the targeted rna from a specific orf would no longer be generated by specific mutation . pk15 cells seeded in 6-well tissue culture plates were transfected with the plasmids using lipofectamine 2000 ( invitrogen ) according to the manufacturer 's instruction . we have designed nine experimental groups , each containing 3 repeats . at 12 post - transfection , total rna was extracted from the cells using trizol ( invitrogen ) following the manufacturer 's recommendations . the quantity and quality of each rna sample were evaluated using a nanodrop spectrophotometer ( thermo scientific , usa ) . the total rna was used to synthesize double - strand cdna and in vitro transcribed to crna , which was used to synthesize 2nd - cycle cdna and then hydrolyzed by rnase h using ambion wt expression kit for affymetrix genechip ( affymetrix , lot . 4011974 , usa ) , and then 2nd - cycle cdna was fragmented and the single - stranded cdna was labeled with wt terminal labeling kit ( affymetrix , lot . 900671 ) and hybridized on porcine gene 1.1 st array strip ( affymetrix , lot . the hybridized arrays were washed and stained on the geneatlas fluidics station ( affymetrix ) . the scanned images were first assessed by visual inspection then analyzed to generate cel files using the default setting of affymetrix geneatlas workstation software . then the raw data ( .cel files ) were normalized and summarized with the affymetrix microarray suite 5.0 ( mas5 ) and with the robust multi - array average ( rma ) algorithm  . in a comparison analysis , a two class unpaired method in the significant analysis of microarray software ( sam , version 3.02 ) was applied to identify significantly differentially expressed genes between test and control groups . q value was set to 0.05 and transcripts were filtered on the basis of   1.2-fold difference . the microarray cel files , normalized data and experimental information have been deposited in ncbi 's gene expression omnibus database under accession number gse71945 . based on hypergeometric distribution , all differentially expressed genes were analyzed using two databases , gene ontology ( go ) and kyoto encyclopedia of genes and genomes ( kegg ) pathway , and statistical significance was calculated when their q - values were <  0.05 . here , we described that functions of the proteins encoded by orfs 1 to 8 were characterized by microarray . among these , by kegg pathway analysis , the pathways involved in the digestive gland ( gastric acid , salivary and pancreatic ) secretion and the neurodegenerative diseases ( alzheimer 's disease and huntington 's disease ) induced by orf1 were the most interesting . a tandem dimmer of p1 genome was cloned into the bamhi site of bluescript plasmid ( invitrogen ) , which was regarded as the  parent  to construct all other p1 mutant plasmids . the targeted rna from a specific orf would no longer be generated by specific mutation . pk15 cells seeded in 6-well tissue culture plates were transfected with the plasmids using lipofectamine 2000 ( invitrogen ) according to the manufacturer 's instruction . we have designed nine experimental groups , each containing 3 repeats . at 12 post - transfection , total rna was extracted from the cells using trizol ( invitrogen ) following the manufacturer 's recommendations . the quantity and quality of each rna sample were evaluated using a nanodrop spectrophotometer ( thermo scientific , usa ) . the total rna was used to synthesize double - strand cdna and in vitro transcribed to crna , which was used to synthesize 2nd - cycle cdna and then hydrolyzed by rnase h using ambion wt expression kit for affymetrix genechip ( affymetrix , lot . 4011974 , usa ) , and then 2nd - cycle cdna was fragmented and the single - stranded cdna was labeled with wt terminal labeling kit ( affymetrix , lot . 900671 ) and hybridized on porcine gene 1.1 st array strip ( affymetrix , lot . the hybridized arrays were washed and stained on the geneatlas fluidics station ( affymetrix ) . the scanned images were first assessed by visual inspection then analyzed to generate cel files using the default setting of affymetrix geneatlas workstation software . then the raw data ( .cel files ) were normalized and summarized with the affymetrix microarray suite 5.0 ( mas5 ) and with the robust multi - array average ( rma ) algorithm  . in a comparison analysis , a two class unpaired method in the significant analysis of microarray software ( sam , version 3.02 ) was applied to identify significantly differentially expressed genes between test and control groups . q value was set to 0.05 and transcripts were filtered on the basis of   1.2-fold difference . the microarray cel files , normalized data and experimental information have been deposited in ncbi 's gene expression omnibus database under accession number gse71945 . based on hypergeometric distribution , all differentially expressed genes were analyzed using two databases , gene ontology ( go ) and kyoto encyclopedia of genes and genomes ( kegg ) pathway , and statistical significance was calculated when their q - values were <  0.05 . here , we described that functions of the proteins encoded by orfs 1 to 8 were characterized by microarray . among these , by kegg pathway analysis , the pathways involved in the digestive gland ( gastric acid , salivary and pancreatic ) secretion and the neurodegenerative diseases ( alzheimer 's disease and huntington 's disease ) induced by orf1 were the most interesting .
porcine circovirus - like agent p1 is a newly discovered virus containing a single - strand circular genome . 
 the genome of p1 is a dna molecule of 648 nucleotides which contains eight open reading frames ( orfs ) that probably encode potential proteins or polypeptides . 
 thus it is very important to clarify these proteins ' function . 
 here we provide the methods and analysis of microarray data in detail to characterize the transcriptome profile of p1 with and without the orf . 
 the relevant microarray data sets have been deposited in gene expression omnibus ( geo ) database under accession number gse71945 .