| Porcine circovirus（PCV）is divided into four genotypes,porcine circovirus type 1（PCV1）,porcine circovirus type 2（PCV2）,porcine circovirus type 3（PCV3）and porcine circovirus type 4（PCV4）.PCV1 is non-pathogenic to swine,whereas PCV2 is a pathogen of porcine circovirus-associated disease,causing great economic losses for the pig production industry worldwide,PCV3is a newly emerging porcine circovirus exhibiting a wide range of clinical syndromes,which may be associated with the pathogenicity observed in pigs,PCV4 was first discovered in China in 2019,and its prevalence and pathogenicity remain unknown.China has the largest swine industry scale and pork consumption worldwide,accounting for more than half of the world.The purpose of this study is to investigate the molecular epidemiology of PCV2 and PCV3 in swine herds in China,to clarify the current infection,genetic diversity and evolution of PCV2 and PCV3 in China,and isolate and identify the pathogenicity of PCV2 epidemic strains.At the same time,based on the sequence of the epidemic strains,we carried out the research of PCV2 and PCV3 recombinant adenovirus vaccine,evaluated the immune effect of the vaccine and the protection effect of the epidemic strains through animal experiments,and laid the foundation for the prevention and control of PCV2 and PCV3 infection.The specific results of this study are as follows:（1）In order to study the molecular epidemiological characteristics of PCV2 in China,3201clinical samples from 206 pig farms in 9 provinces of China from 2016 to 2019 were evaluated by PCR.It was shown that the overall prevalence of PCV2 infection was 39.6%（1269/3201）,from18.6%-44.7%in different provinces;the overall prevalence of PCV2 infection was 84.5%（174/206）at the farm levels,suggesting that PCV2 infection is prevalent in China.To further study the genetic variation and evolution of PCV2,we sequenced the entire genome of 64 PCV2 strains and an additional 178 ORF2 sequences.The homology of the whole genome sequence,ORF1 and ORF2genes are 93.9%-99.9%,96.6%-100%and 86.8%-100%respectively;By analyzing PCV2 strains,126 and 96 amino acid mutations were observed among Cap and Rep respectively,indicating the high genetic variation of PCV2 strains.Recombination analysis showed that different PCV2 strains could recombine in pigs to produce new strains.Phylogenetic analysis revealed that all isolates belonged to PCV2a,PCV2b and PCV2d,among them,18 belongs to PCV2a,59 belongs to PCV2b and 190 belongs to PCV2d,indicating that the PCV2d genotypes currently prevalent in pigs from China.（2）Based on the analysis of global strain sequences,the visualized genotype drift and amino acid variation on the evolution of PCV2 were determined,and the possible causes and effects of amino acid mutations were analyzed.The visualized genotype drift during the evolution of PCV2worldwide shows that,before 2001,PCV2a was the main genotype,from 2001 to 2011,PCV2b was the main genotype,and after 2011,PCV2d was the main genotype;after 2006,an increasing PCV2d and decreasing PCV2a and PCV2b was showed;PCV2 experienced two genotype drifts during its evolution.In 2001,PCV2a drifted to PCV2b,and in 2012,PCV2b drifted to PCV2d.In China,before 2002,PCV2a was the main genotype,from 2002 to 2008,PCV2b was the main genotype,after 2008,PCV2d was the main genotype;after 2004,an increasing PCV2d and decreasing PCV2b was showed;Two genotype drifts occurred on the evolution of PCV2.In 2002,PCV2a drifted to PCV2b,and in 2009,PCV2b drifted to PCV2d.During the evolution of the PCV2,14 amino acid mutations on the Cap changed significantly,and the occurrence time was around 2007.The PCV2commercial vaccine was used worldwide in 2006,indicating that the vaccine is used and immune pressure may cause amino acid mutations.Since the vaccines used before 2015 were all PCV2a genotypes,further analysis of the amino acid composition of the PCV2a,PCV2b,and PCV2d genotypes revealed that the Cap protein amino acid variation was mainly determined by the PCV2a and PCV2b genotypes,and after the mutation was determined by the PCV2d genotype.It shows that the use of vaccine may result in amino acid mutation in PCV2 during the evolution process,and amino acid mutation will lead to the emergence and prevalence of PCV2d genotype.（3）The molecular epidemiological investigation results of PCV2 indicate that the PCV2strains currently prevalent are dominated by the PCV2d genotype.To further determine the PCV2epidemic strain,the PCV2d Cap protein amino acids were compared,and it was found that the PCV2/JL/JL19 strain Cap protein sequence is conservative,and there is no amino acid variation compared with other strains,so it is determined to be the dominant PCV2 strain.To clarify the pathogenicity of the PCV2/JL/JL19 epidemic strain,the virus was isolated and the virus titer could reach 106.0 TCID50/m L.In the swine body challenge test,the clinical symptoms mainly showed decreased appetite and rough back hair,and the body weight decreased significantly 28 days after the challenge;the clinical anatomy showed that the lesions were mainly lymphadenopathy and bleeding,and high levels could be detected in various tissues and organs.The copy number of PCV2DNA produces obvious viremia;histopathological observation of inguinal lymph nodes and lungs produces obvious pathological damage.These results indicate that the current PCV2 epidemic strains are significantly pathogenic to swine.（4）In order to study the molecular epidemiological characteristics of PCV3 in China,4199clinical samples from 278 pig farms in 10 provinces of China from 2016 to 2019 were evaluated by PCR.It was shown that the overall prevalence of PCV3 infection was 29.4%（1235/4199）,from7.3%-37.3%in different provinces;the overall prevalence of PCV3 infection was 74.8%（208/278）at the farm levels,suggesting that PCV3 infection is prevalent in China.Furthermore,a statistical analysis showed PCV3 might exacerbate PCV2 and PRRSV infection rate and have a potential association with pig clinical disease.In addition,we sequenced the entire genome of 70 PCV3strains;homology analysis showed that PCV3 strains have more than 96%similarities at the nucleotide acid level,and PCV3 shares less than 60%similarities with other circoviruses.By comparing the total 673 PCV3 strains from the NCBI Gen Bank,we found the major of amino acid mutations are located in predicted epitope regions and the mutations ratio changed during PCV3evolution.Phylogenetic analysis revealed that all isolates belonged to PCV3a and PCV3b,and increasing PCV3a and decreasing PCV3b trends were observed during PCV3 evolution.Overall,this study provides important insights for understanding PCV3 prevalence,pathogenesis,and evolution and will guide future efforts to develop effective preventive and control measures.（5）Epidemiological survey results show that PCV3 has a high positive rate in the pig herds of China.Apart from transmission among pigs,is there other possible transmission routes,such as vectors from mosquitoes.In order to understand the infection and genomic characteristics of PCV3in mosquitoes,herein,269 mosquito samples from pig farms were collected in Heilongjiang,Jilin and Yunnan provinces and PCV3 detection was done by PCR.It was shown that the total PCV3-positive rate in mosquitoes was 32.0%（86/269）,ranging from 21.4%to 42.5%at the pig farms,implying that mosquitoes may serve as a route of transmission for PCV3.To determine the possible origin of PCV3 in mosquitoes,80 pig serum samples were collected on those pig farms with captured mosquito samples.The PCV3-positive rate in pig farms ranges from 15.0%to 30.0%,and the positive rate was positively correlated between mosquitoes and pigs.Additionally,we sequenced the entire genome of 6 strains of PCV3 in mosquitoes and 2 strains of PCV3 in pigs.Sequence analysis indicated that 100%nucleotide similarity of PCV3 between mosquitoes and pigs from the same pig farm.Phylogenetic analysis showed that PCV3 could be divided into two clades,PCV3a and PCV3b,and the PCV3 strains isolated in mosquitoes were distributed on the two clades.These results demonstrate that mosquitoes may serve as potential transmission vector in the life-cycle of PCV3,revealing possible transmission routes of PCV3.（6）Analysis of the registration data of the novel veterinary drug of the PCV2 vaccine and the data of clinical trial approval show that the PCV2 genetically engineered vaccine and the PCV2-based dual vaccine are the development trend of the PCV2 vaccine.PCV3,as a novel infectious disease in swine,is a member of the circovirus family of PCV2 in the same way as PCV2.It is related to various clinical symptoms of swine and brings great harm to the swine industry.With reference to the Cap amino acids of PCV2 epidemic strain PCV2/JL/JL19 and PCV3 epidemic strain PCV3/JL/JL32 obtained in molecular epidemiological studies,the codon optimization for pig species was carried out applying adenovirus as a vector to obtain single expression recombinant adenoviruses r Ad5-PCV2-Cap and r Ad5-PCV3-Cap of PCV2 Cap protein and PCV3 Cap protein;and recombinant adenovirus r Ad5-PCV2-Cap-PCV3-Cap expressing both PCV2 Cap protein and PCV3 Cap protein.Sequencing showed that the genes were correct,Western Blot and IFA identified proteins were expressed,and stability experiments showed that the three recombinant adenoviruses had good stability,indicating that the recombinant adenovirus was successfully constructed.（7）The mouse immunization test showed that the recombinant adenovirus r Ad5-PCV2-Cap and r Ad5-PCV2-Cap-PCV3-Cap vaccine immunized 35 days later,PCV2-specific antibody titers can reach 1:2700 and 1:2100;and neutralizing antibody are 1:43 and 1:52 respectively;lymphocyte proliferation levels are 1.37 times that of the control group PBS;both can produce higher levels of cytokines IFN-γand IL-4,causing Th1 and Th2 type immune responses.Recombinant adenoviruses r Ad5-PCV3-Cap and r Ad5-PCV2-Cap-PCV3-Cap vaccine immunized pigs 35 days later,PCV3-specific antibody titers were up to 1:2100 and 1:1600;lymphocyte proliferation levels were in the control 1.3 times of PBS;both can produce higher levels of cytokines IFN-γand IL-4,causing Th1and Th2 type immune responses.These results indicate that recombinant adenovirus vaccines r Ad5-PCV2-Cap and r Ad5-PCV3-Cap can immunize mice to produce better humoral and cellular immune responses of PCV2 and PCV3,respectively;After immunization of mice,they can simultaneously produce better humoral and cellular immune responses of PCV2 and PCV3.（8）Swine body immunization test showed that the recombinant adenovirus vaccines r Ad5-PCV2-Cap and r Ad5-PCV2-Cap-PCV3-Cap 35 days after immunization,PCV2-specific antibody titers can reach 1:1300;neutralizing antibodies is 1:85 and 1:81 respectively;the lymphocyte proliferation level is 1.46 times and 1.4 times that of the control group PBS;both can produce higher levels of cytokines IFN-γand IL-4,causing Th1 and Th2 type immune responses.After the PCV2strain PCV2/JL/JL19 was challenged,the recombinant adenovirus vaccines r Ad5-PCV2-Cap and r Ad5-PCV2-Cap-PCV3-Cap immunized group had no obvious clinical symptoms compared with the control group,which could reduce the number of viremia pigs.There was no obvious pathological damage to the lung and inguinal lymph nodes.Recombinant adenovirus vaccines r Ad5-PCV3-Cap and r Ad5-PCV2-Cap-PCV3-Cap immunized pigs 35 days later,PCV3-specific antibody titers were up to 1:1600 and 1:1300,respectively;lymphocyte proliferation levels were respectively1.35 times and 1.32 times that of the control group PBS;both can produce higher levels of cytokines IFN-γand IL-4,causing Th1 and Th2 type immune responses.After PCV3 tissue challenge,the recombinant adenovirus vaccines r Ad5-PCV3-Cap and r Ad5-PCV2-Cap-PCV3-Cap immunized group can reduce the number of PCV3 viremia pigs compared with the control group,and the lung and inguinal lymph nodes have no obvious pathology damage.These results indicate that the recombinant adenovirus vaccines r Ad5-PCV2-Cap and r Ad5-PCV3-Cap can produce humoral and cellular immune responses against PCV2 and PCV3,respectively,which can resist the attacks of PCV2 and PCV3,and provide the swine body with excellent protective effect;recombinant adenovirus vaccine r Ad5-PCV2-Cap-PCV3-Cap can produce humoral and cellular immune responses against PCV2 and PCV3 at the same time,and resists the attacks of PCV2 and PCV3. |
