The Complete Genome Sequencing And Analysis Of Porcine Circovirus Type 2 Strains From Anhui

Since 1991,the clinical infection of porcine circovirus type 2(PCV2)is becoming more and more serious.It has been widely spread all over the world,and has caused great economic losses to the pig industry all over the world,which has seriously restricted the development of pig industry.PCV2 is associated with many clinical diseases,such as PMWS,PDNS,PRDC,reproductive disorders of sows and CT.These diseases are collectively called PCVD.PCVD mainly adopts comprehensive control measures,including feeding management,biosafety,vaccine immunization and drug health care,among which vaccine immunization is the most effective method to prevent and control PCVD.In recent years,the genotype of PCV2 epidemic strains has gradually changed from PCV2b to PCV2d at home and abroad.However,whether commercial vaccines based on PCV2a and PCV2b have protective effects on PCV2d infection has attracted the attention of scholars all over the world.The purpose of this study is to understand the genetic variation of PCV2 strains in Anhui Province,so as to lay a foundation for the study of molecular epidemiology and source of PCV2 strains,accumulate effective materials for the development of new PCV2 vaccine,and provide a basis for further research and scientific prevention and control of PCVD in the future.In this study,15 PCV2 Anhui isolates strains were used to proliferate by PK-15 cells,then detected by PCR and observed by electron microscopy.According to the genome sequence of PCV2 published by GenBank,two pairs of specific primers were designed.Then,15 PCV2 Anhui isolates strains were cloned and sequenced by PCR.The nucleotide sequence and the ratio of deduced amino acids of whole genome and main open reading frames(ORF1,ORF2)was analyze by DNA STAR software.Genetically Developmental Evolution Tree Mapping Nucleotide Sequences Through MEGA7.0 Software.At the same time,15 PCV2 Anhui isolates strains were compared with 31 PCV2 reference strains in GenBank at home and abroad.The results showed that:(1)The similarity of the whole genome nucleotide sequence of 15 PCV2 Anhui isolates strains was 95.4%-99.8%.The similarity of ORF1 nucleotide and deduced amino acid sequence was 96.6%-100%,while the similarity of ORF2 corresponding sequence was 93.2%?100%.Compared with 19 reference strains in China,the similarity of whole genome sequence was 92.9%?99.8%,the similarity of ORF1 nucleotide and deduced amino acid was 96.3%?99.9%,and the similarity of ORF2 corresponding sequence was 91.5%-99.9%.Compared with 12 reference strains from abroad,the similarity of the whole genome sequence was 55.0%?99.8%.The similarity of the whole genome sequence with NO.38 from the US was 55.0%-55.3%.The similarity with N0.39 from the UK was 77.0%-77.9%.The similarity with other 10 reference strains was 94.3%-99.5%.The similarities of ORF1 nucleotides and deduced amino acids were 96.4%-99.8%,and those of ORF2 sequences were 89.9%?96.4%.(2)According to the phylogenetic tree,The genetic distances of 9 PCV2 Anhui isolates strains(Nos.3,6-13)and 5 domestic reference strains(Nos.17,21,24,25,29)and 2 foreign reference strains(Nos.43,44)were relatively close,belonged to the same PCV2d genotype.The genetic distances of 6 PCV2 Anhui isolates strains(Nos.1,2,4,5,14,15)and 12 domestic reference strains(Nos 18,19,22,23,26,27,28,30,31,32,33 and 34)and 3 foreign reference strains(36,40 and 46)were relatively close,belonging to the PCV2b genotype.No strains of PCV2c and PCV2e were found.Conclusion:(1)The homology of genome-wide and main open reading frames(ORF1,ORF2)nucleotides and deduced amino acid sequences among PCV2 Anhui isolates strains was high,with similarities of 93.2%-100%.The prevalence of PCV2 in Anhui has no obvious regional difference.The similarity between PCV2 Anhui isolates strains and domestic reference strains was 91.5%-99.9%.There is no significant regional difference in the prevalence of PCV2 in China.The similarity between the isolates and the reference strains abroad is 55.0%-99.8%,which is lower than that of the reference strains at home.The prevalence of PCV2 shows some regional differences.(2)15 PCV2 Anhui isolates strains had close genetic relationship with each other and had little genetic variation.Nine of them belonged to PCV2d gene subtype and six belonged to PCV2b gene subtype.The genotype of PCV2 epidemic strains in Anhui Province had changed from PCV2b to PCV2d.At present,PCV2d is the dominant genotype,which is consistent with the relevant reports in many areas in China.