Analysis Of The Genetic Evolution Of PCV2 In Parts Of Hunan And The Difference Of In Vitro Replication Among Distinct Genotypes

Porcine circovirus type 2（PCV2）causes porcine circovirus associated disease（PCVAD）,which is widespread throughout the country,causing substantial economic losses to the swine industry.PCV2 presents a high frequency of mutations under the selection pressure,which leads to the generation and prevalence of new genotypes,increases the difficulty of clinical virus clearance.To understand the PCV2 prevalence in partial areas of Hunan province and the properties of evolution and mutation,and analyze the difference of in vitro replication among distinct strains,the main results of this study are as follows:1.Molecular epidemiological investigation of 223 tissue samples collected from14 regions in Hunan Province showed that the positive rate of PCV2 was 51.27%（115/223）,and co-infection between different pathogens is common,among which the co-infection rate of PCV2 and porcine parvovirus（PPV）,PCV2 and porcine reproductive and respiratory syndrome virus（PRRSV）were the highest,reaching16.52%（19/115）.Phylogenetic tree analysis based on the complete genomes of 43PCV2 strains from the positive samples showed that 6.98%（3/43）of the strains were PCV2a,53.49%（23/43）were PCV2b and 34.88%（15/43）were PCV2d.In addition,two strains were identified as the recombinants between PCV2b and PCV2d by using homologous analysis.2.Based on the protein primary structure comparison and three-dimensional structure analysis,Loop DE,Loop FG and the NLS regions of PCV2 capsid protein subunit（Cap）are relatively conserved,while Loop BC,Loop CD,Loop GH,and Loop HI are the hot spots of mutation.Combined with the PCV2 capsid surface structure,with the evolution of epidemic strains from PCV2a to PCV2b,and then to PCV2d,the number of positively charged amino acids in the peak areas（include the five-fold axes,Loop EF and the carboxyl termini）increases gradually,it is speculated that it is beneficial for viruses binding to the negatively charged receptor（such as heparan sulfate and chondroitin sulfate B）.In-depth analysis of the key amino acid mutation sites of the Cap,some amino acid mutation sites located in the glycosaminoglycans（GAGs）receptor binding regions（such as 59,63,68,134,169）and neutralizing antibodies recognition sites（such as 59,131,151,190,191）.PCV2Rep protein is relatively conserved,and only several sites showed high mutation rates,but the effects of the mutations on the biological function of the virus need to be further studied.3.Six infectious cloning plasmids include PCV2a-LY1908,PCV2b-YY1908.3,PCV2b-CD1922,PCV2d-YY1908.1,PCV2d-WZ1901.4（were constructed using the isolate strains obtained in this study）and PCV2a-KJ437192（previously constructed by our team）,which include PCV2a,PCV2b and PCV2d,were used for virus rescue to study the differences of in vitro replication among these strains with distinct genotypes.The results showed that the infectious clones of strains PCV2b-CD1922,PCV2d-YY1908.1 and PCV2d-WZ1901.4 were successfully rescued via their corresponding infectious DNA clones and stably passaged in PK-15 cells,strains PCV2b-YY1908.3 and PCV2a-KJ437192 could rescue the virus,while the genomic copy numbers of the rescued virus decreased during consecutive passages,and finally,strain PCV2a-LY1908 could not be rescued from its infectious DNA clones.Among the rescued viruses,the virus titers of PCV2b-CD1922 and PCV2d-YY1908.1 were both higher than 10⁵ TCID₅₀/ml,and used in subsequent studies of apoptosis and replication kinetics.The results demonstrated that both viruses could induce apoptosis,but there is no significant difference between them,and both strains in vitro replicate very well,while the replication ability of strain PCV2d-YY1908.1 was higher than that of strain PCV2b-CD1922.