Recombinant Plasmid Construction Of Porcine Circovirus Type 3 And Genetic Evolution Analysis

Porcine circovirus type 3(PCV3)is a new type of virus discovered in 2015.At present,PCV3 is prevalent in the world.Sows with reproductive disorders are frequently infected with the virus,and co-infection occurs when the virus can be detected in certain disease.PCV3 has caused certain economic losses to the global pig industry.PCV3 has not been successfully isolated and cultured in passaged cells so far.In order to reduce the loss of PCV3 to China's pig industry and provide support for the study of its pathogenic mechanism,the whole genome sequence of PCV3 of Hebei strain(GenBank accession number:MK105924)was used to construct infectious clone,which was then transfected into the passage cells for proliferation.In order to further understand the structural characteristics,pathogenic mechanism and the relationship between the distribution and the region of PCV3,the sequence analysis of Hebei strain and the global PCV3 genome will provide the basis for the prevention and control of the virus.A new strain of PCV3 was found in the disease material collected in North China.In order to amplify the entire genomic sequence of this new PCV3 strain,two pairs of primers were used to amplify it.After sequencing the genome of the obtained product,the whole genome sequence of PCV3 was obtained and named as Hebei strain PCV3.An infectious clone was constructed from this whole genome sequence,and a recombinant plasmid was obtained.The constructed recombinant plasmid was transfected into PK-15 cells and passaged on continuously for 15 generations.The genetic map of the Hebei strains was drawn and 9 PCV3 strains were selected from Hebei Province.These 10 genomes were compared and analyzed.The genetic evolution relationship was analyzed by software and the genetic evolution tree was drawn,the analysis was also carried out on 176 global strains to be published on GenBank in 2019.The genotype of Cap protein in Hebei strain was 3b subtype,and its homology with other PCV3 Cap protein strains in Hebei province was between 97.8%and 99.8%.The homology of the whole genome sequence of the obtained Hebei strain PCV3,PCV1 and PCV2 was less than 50%,the genetic evolution tree analysis showed that the three PCVs were in different branches,indicating that they belonged to different genotypes.The homology with the whole genome sequence of other 43 PCV3 strains at home and abroad was between 98.7%-99.3%,and the highest homology can reach 99.3%,this is a PCV3 strain from Russia(GenBank accession number:MG679916),they are the closest relatives,it can be seen that the whole genome sequence of different PCV3 strains has a high homology,and no significant variation has been found.The genetic evolution analysis of the whole genome sequence of 175 PCV3 strains in the world revealed that the number and distribution of PCV3 subtypes were related to the region.Conclusion:A new PCV3 strain(GenBank accession number:MK105924)was obtained as the 3b subtype.PCV3 infectious clone was successfully constructed,and was continuously transmitted 15 generations.There are regional differences in the distribution of PCV3 and its subtypes.