| Objectives Porcine circovirus type2(PCV2) is the primary causative agent of postweaningmultisystemic wasting syndrome (PMWS), which causes significant economic losses to theswine industry. PCV2is also a potential zoonotic virus; however, the pathogenesis of PCV2infection is still unclear. With the molecular pathogenesis research of PCV2, three open readingframes (ORFs) have been detected and characterized: ORF1encodes Rep and Rep’ proteins,which are nessary for viral replication; ORF2encodes the viral capsid protein; ORF3encodesapoptosis associated protein. ORF4overlaps completely in ORF3, and the function of this geneis still unknown. In this work, ORF4-deficient PCV2mutants were produced for its function inthe viral replication and the virus-infected apoptosis. The transcription of ORF3and ORF4hasbeen detected, and two ORF3/ORF4associated3′poly (A) sites were found in previously work,however, their5’ initiation sites were not obtained yet. This study lays the foundation for furtheranalysis of the ORF4gene and provides new methods and theoretic basis for investigating thepathogenesis of PCV2.Methods Firstly, on the basis of the sequenced PCV2genomic DNA, mutational sites wereselected by computerized predictions. The start codon or in-frame termination mutation wasengineered. Mutant plasmids were constructed according to the principle of the site-directedgene mutagenesis kit, the full-length mutant PCV2genomes were excised from plasmids byrestriction enzyme. After recircularized by T4DNAligase, the ligated viral DNAwas transfectedinto confluent PK-15cells, so as to produce the mutant PCV2viruses. The abilities of viralreplication, specific RNA donor splice junction and the expression of viral caspid were detected,to make sure the infectivity of the progeny viruses. Secondly, according to the sequenced PCV2strain, three pairs of specific primers were designed to establish real-time quantitative PCRsystems, which were used for virus titration and quantification of the mRNAexpression of ORF1,ORF3and ORF4. The differential expressions of viral DNA or RNA in wild-type, recombinant,or mutant PCV2viruses-infected cells would provide the evidences to analyse whether the ORF4affects the replication of PCV2in PK-15cells. Cellular apoptosis was evaluated by measurementof caspase activity between recombinant and mutant PCV2, to investigate the viral function ofvirus-induced apoptosis. Lastly,5’ RACE experiments were carried out to determine the5’initiation site of the ORF4mRNA.Results (1) Infective recombinant and mutant viruses were constructed successfully, and themutantion can still be detected in15th generation progeny virus;(2) The Real-time PCR assayswere successfully developed and had a linear quantification range of2.53×102~2.53×107copies DNA for virus titration and quantification of the mRNA expression of ORF1, ORF3, or ORF4(R2>0.99);(3) PK-15cells were infected by equal titer of wild-type, recombinant, or mutantPCV2, and the copies of viral DNA and the mRNA expression of ORF1, ORF3, or ORF4wereanalyzed. The result showed that ORF4was not essential for viral replication. The deficiency ofORF4protein was found to enhance the expression of ORF3mRNA which expresses aapoptosis-associated protein; the ORF4-deficient virus also contributed to the more active viralreplication capacity and shown a higher expression of ORF1at transcription level than wild-typePCV2.(4) Cellular apoptotic evaluation suggested that the deficient-ORF4PCV2triggeredhigher activities of caspase-3and caspase-8than wild-type virus;(5) Anew5’ initiation site at nt593was detected by5’ RACE, and was matched to the3’ poly (A) site at nt246. This new foundtranscript is produced from ORF4. |
