Molecular Epidemiology Of Porcine Circovirus Type 2 And Immune Characterization Of Cap Protein

PCV2 proliferation in the lymphatic system can cause immunosuppression,and induce secondary infection and mixed infection of other pathogens,such as prorcine parvovirus,pseudorabies virus,streptococcus suis,etc.Since the end of the 1990 s,PCV2has become the main pathogen of pigs in the world.PCV2 mainly infects pigs aged4-16 weeks and presents as multiple system failure syndrome(PMWS),procine dermattis nephrotic syndrome(PDNS),reproductive disorders and respiratory diseases,has caused great harm to the global swine industry.The study found that the whole genome of PCV2 contains 11 ORF(Open reading frames).The gene of ORF2 mainly encodes the nucleocapsid protein related to the virus structure,namely the Cap protein,which is also an important immunogenic protein of the virus and can induce the body generate neutralizing antibodies against PCV2.According to the international classification method based on the proportion of different nucleotide sites in ORF2,five main PCV2 genotypes were identified: PCV2 a,PCV2b,PCV2 c,PCV2d and PCV2 e.Several studies have shown that PCV2 b is highly pathogenic and is the main gene subtype prevalent in the early stage of China,Succential studies showed that a dynamic genotype shift occurs from PCV2 b to PCV2 d and that PCV2 d appears to dominate in the worldwide.The most economical and effective way to prevent and control circosis in pigs is immunization with highly specific vaccine.Currently,vaccines on the market are basically developed based on PCV2 a and PCV2 b,with insufficient protection against PCV2 d.There may be recombination between PCV2 genotypes.Therefore,it is of great importance to study the molecular epidemiology of PCV2.To explore the prevalence of PCV2 in pig farms in central and southern China from 2019 to 2020,the homologous and heterologous protection ability of PCV2 b and PCV2 d Cap proteins based on the baculovirus against the PCV2 b and PCV2 d infections was evaluated.Reference for PCVAD prevention,control and development of PCV2 vaccine in China.1707 suspected PCV2 samples from 414 large-scale pig farms were detected by quantification PCR(q PCR).Meanwhile,the 50 PCV2 positive samples were randomly selected from seven provinces and the ORF2 gene was amplified by PCR,and isolated by gel extraction.Then,the ORF2 gene was sequenced and analyzed by Meg Align and MEGA6 for phylogenetic analysis.The recombinant baculovirus of PCV2 b and PCV2 d Cap proteins were constructed by the Bac-to-Bac Baculovirus Expression System(Invitrogen,USA)following the manufacturer’s instructions.The recombinant baculovirus titers were computed by immunoflfluorescence assay.Sf9 cells were infected with rBac-2b Cap and rBac-2d Cap and then harvested post-infection(dpi),the purifified sample was assembled into virus-like particles(VLPs).With the Safety of Animal Immunization Therefore,the homologous and heterologous protection ability of PCV2 b and PCV2 d Cap proteins based on the baculovirus against the PCV2 b and PCV2 d infections was evaluated,which is expected to design and develop excellent PCV2 protein vaccine candidatesThe results showed that the PCV2 Positive rate of 1707 samples was 36.08%(616/1707),and 49.03%(203/414)farms were infected.The PCV2 infecting rate of lung was 41.31%,fetus was 34.51%,blood sample was 31.97%,semen was 4.12%,respectively.The nucleotide homology among ORF2 genes from 50 PCV2 strains was84.1% ～100%,and its range was from 75.6% to 99.8% comparing to 18 reference sequences.Genetic evolution analysis showed that PCV2 d accounted for 80%(40/50),PCV2 a for 4%(2/50),PCV2 b for 16%(8/50).However,PCV2 c and PCV2 e were not detected.Among them,the PCV2 d was accounts for the main genotypes in Henan,Hubei,Hunan,Sichuan,Shandong,Yunnan and Guangdong province.The main epidemic genotype of PCV2 in Central and southern China from 2019 to 2020 is PCV2 d,followed by PCV2 a and PCV2 b.The main prevalent genotypes of PCV2 in central and southern China from2019～2020 were PCV2 d,followed by PCV2 a and PCV2 b.PCV2b Cap and PCV2 d Cap have some cross-protection effects against PCV2 b and PCV2 d strains.Therefore,this study provides a theoretical basis for the development and clinical prevention and control of PCV2 vaccine.The results provided the certain guidance for the research and development of PCV2 vaccine and control of PCV2 disease.