Study On The Co-infection Among Different Genotypes And The Evolution Of Porcine Circovirus Type 2(PCV2)

Porcine circovirus(PCV),a small icosahedral nonenveloped virus with a single-stranded circular DNA genome,belongs to the circoviridae family and is one of the smallest viruses currently found in the mammals.Porcine circovirus type 2(PCV2)is closely associated with postweaning multisystemic wasting syndrome(PMWS)and other syndromes which collectively are called PCV related diseases(PCVRD).The infection of PCV2 is widespread and serious consequence,thereby causing significant economic losses in the swine industry worldwide.This virus has two main Open Reading Frames(Open Reading Frames,ORFs),mainly responsible for coding replication proteins and structural protein,respectively.Additionally,there is a repetitive sequence between the two major open reading frames of the genome,and the virus genome of circoviridae has stem loop structure,participating in the rolling circle replication.In the present study,viral genomic DNAs were extracted from various frozen tissues(lymph node,spleen,and lung,respectively)of the individual diseased pig in a field of Yiyang,Hunan.The aforementioned DNA samples were used as templates to amplify the whole PCV2 genome by PCR.Following bacterial transformation,twenty-three clones were randomly picked-up and plasmid DNAs were prepared.Finally,the 69 clones containing PCV2 genomic DNAs were sequenced.The genome sequences of the new isolates were confirmed via the online BLAST program,after which the sequences of these novel strains were submitted to the NCBI database.Furthermore,based on the genome sequences of the 11 PCV2 strains,a phylogenetic tree was constructed using MEGA5.05 with Kimura-2 parameter model,and the result clearly demonstrated the 11 strains readily fitted into two clusters of 1B and 1C within the PCV2 b branch.Importantly,we found some special strains: YiY-3-2-11 and YiY-3-2-3.The strain YiY-3-2-11 might be an intermediate,representing a subtype switch of 1B to 1C through a key mutation in the 3' termini of ORF2.Another mutant derived from the strain of YiY-3-2-11,contains a point mutation causing the replacement of UAG(stop codon)with AAG,resulting in an extra lysine present at the carboxyl terminus of the Cap protein in the strain YiY-3-2-10.These findingssuggest the existence of different strains of PCV2 b in an individual may not only come from co-infection,but may be due to additional point mutations acquired in the course of immune evasion.In addition,one strain,designated as YiY-3-2-3,had a point mutation(G710A)in ORF1,leading to a shortened rep gene,that encodes a carboxy-termini truncated Rep protein(315 versus 219 aa).Accordingly,the effects of this mutant on the biological functions of the Rep protein and viral DNA replication need to be further determined.In order to investigate the effects of the shortened ORF1 area on the PCV2 replication and the PCV2 viral load on the PCV2 infectious ability,infectious clones and rescued viruses were constructed.Three kinds of virus genomes were inserted into pSP72 vector to construct double copies infectious clones,pSP72-IC-YiY-3-2-1,pSP72-IC-YiY-3-2-3 and pSP72-IC-YiY-3-2-10,by using gene clone techniques.These three infectious clones were then transfected into PK-15 cells,and analyzed by real-time PCR and IFA.All these results showed that the three infectious clones can produce rescued virus.Additionally,the results indicated that there is a significant difference between these three infectious clones,the shortened ORF1 area has obviouseffects on PCV2 replication.Furthermore,PCV2 viral load was positively correlated with PCV2 infection.The present study not only provides the basis for clinical test of PCV2 and pathogen of genetic variation analysis,but also lay a solid foundation for the pathogenicity of the virus.