Whole Genome Amplification And Construction Of Infectious Clone Of PCV2 Genome Of Hunan Strain

PCV, short for Porcine circovirus, as a member of Circoviridae family, is the smallest mammalian virus in present. There are two main kinds of PCV2 genotypes or subtypes,which are PCV2a and PCV2b. Current research suggests that,the varition of PCV2 has speed up and PCV2b has gradually became the dominant strain. It is a common thing on clinic that multiple PCV2 subtypes co-infection with each others and PCV2 has mixed infection with multiple pathogens. PCV2 presents global distribution which can damage the pig’s immune system and lead to severe immunosuppression of pig, thus cause major economic losses for pig breeding.In this study, PCR detection and sequence analysis of PCV2 DNA was used for thirty-one samples of diseased pigs in a certain area of Hunan Province at first, the selected HNLYYA1 as a PCV2 capsid protein amino acid sequence mutant was used to the establishment of MPRCA technical system. Then by the use of the combination of specific primers and random primers, multiply-primed rolling--circle amplification(MPRCA) reaction was used for the detection of total DNA of the PCV2 mutant-HNLYYA1, and at last, the MPRCA products were identified by restriction enzyme of EcoR I and Nco I, PCR, and sequencing reaction.The results show that the size of the digested MPRCA products was the same with PCV2 genomes or fragments. The MPRCA products were cloned into Psp72 vector, then recombinant plasmid was transformed into bacteria DH5a. Finally, the extracted recombinant plasmid was sequenced and sequence alignment analyzed with other sequences. The whole genome was submitted to GenBank with registry number of KJ867555. Sequence alignment and phylogenetic analysis revealed that the genome sequence of PCV2 strain HNLYYAI consists of 1767 nucleotides, its genotype was PCV2b-1C. There is an unique amino acid mutation of PCV2 cap protein when it was aligned with sequences of other strains. The 34th histidine was mutated into tyro sine in NLS, the 169th arginine was mutated into glycine in LOOP GH, and the 206th lysine was mutated into isoleucine in LOOP HI. Later, by the use of the single enzyme of EcoR I and reconnected cyclization, the PCV2 full length cyclization genome was obtained from the MPRCA products of PCV2 strain HNLYYAI. And the genome was transfected into PK-15 cells for subculture and in the end, to complete virus rescue for the virus infectious clones of PCV2 strain HNLYYA1.This study successfully established the technical system of PCV2 MPRCA, revealed the characteristics of complete genome of PCV2 HNLYYAI in Hunan strain, and constructed the PCV2 infectious clone by cell transfection. All the results of this study provide the sequence information base for the evolution of PCV2 strains, and also provide experimental basis for the construction of PCV2 infectious clone, the replication of PCV2 strains and pathogenesis studies. What’s more, the establishment of PCV2 MPRCA technical system also provide a new technical approach for the research for the isolation of complete genomes of circovirus and the construction of infectious clones.