| Porcine circovirus(PCV)is divided into four genotypes:PCV1,PCV2,PCV3,and PCV4.PCV1 is a cell culture contaminant that is not pathogenic to pigs;PCV2 is a globally prevalent pathogen that causes significant economic losses to the swine industry worldwide;PCV3 is a virus discovered in the United States in 2016,which often causes reproductive disorders in pigs and is widely spread globally;PCV4 was first discovered in China in 2019,and its clinical symptoms and pathogenesis are still under investigation.Studies have shown that when pigs are infected with a single PCV subtype,severe PCVD symptoms do not occur,but co-infection between PCV2 and PCV3 can exacerbate clinical symptoms.Therefore,the development of a combination vaccine that simultaneously immunizes against PCV2 and PCV3 is of great significance to production.This study first conducted a molecular epidemiological investigation of PCV2 and PCV3 in pig populations in the southwestern region of China,exploring the infection status in this area.Based on the phylogenetic analysis of the two viruses,the current circulating strains and their genetic variation characteristics were identified.At the same time,a PCV2,PCV3 and IL-4 recombinant pseudorabies virus vaccine was developed,and its biological characteristics were investigated.The safety,immunogenicity,and protective efficacy against viral challenge were evaluated through mouse experiments,laying a foundation for the prevention and control of PCV2 and PCV3.The specific results of this study are as follows:1.Molecular epidemiological characteristics of PCV2 and PCV3 in Sichuan Province,China from 2020 to 2022In order to understand the molecular epidemiological characteristics and genetic diversity of PCV2 and PCV3,this study detected 257 suspected PCVAD pig samples from 23 pig farms in Sichuan Province from 2020 to 2022.The results showed that the positive rates of PCV2 and PCV3 were 26.46%(68/257)and 33.46%(86/257),respectively,and the co-infection rate of PCV2 and PCV3 was 8.95%(23/257),indicating that PCV2 and PCV3 are widely present in the pig population in Sichuan Province.To further study the genetic variation and evolutionary patterns of PCV2 and PCV3,full-genome sequences of some positive clinical samples were amplified,and a total of 12 PCV2 and 18 PCV3 fullgenome sequences were obtained.Homology analysis showed that the homology of PCV2 strains was 94.7%-99.9%,while PCV3 had higher genetic stability,with a homology of 98.7%-99.9%.By comparing with reference sequences in the NCBI database,amino acid mutations of PCV2 were mostly found in its antigenic epitope regions,while mutations of PCV3 were found in both B-cell antigenic epitope regions and antibody recognition regions.Genetic evolution analysis showed that PCV2 strains were divided into three types:PCV2a,PCV2b,and PCV2d,with PCV2d being the most prevalent(53.85%,7/13);PCV3 strains were divided into two types:PCV3a and PCV3b,with PCV3a being the majority(73.68%,14/19).These results provide a theoretical basis for further exploring the epidemiological characteristics,evolutionary patterns,and control measures of PCV2 and PCV3.2.Construction and Partial Biological Characterization of Recombinant StrainsIn this study,two recombinant strains rPRV XJ-ΔgE/gI/TK-2Cap-3Cap and rPRV XJΔgE/gI/TK-2Cap-3Cap-IL4 were constructed.The following abbreviations are for rPRV2Cap/3C ap and rPRV-2Cap/3C ap/IL4.The morphological difference of rPRV-2Cap/3Cap,rPRV-2Cap/3Cap/IL4 and PRV XJ in BHK-21 cells was small by transmission scanning electron microscopy.rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 were continuously passaged in BHK-21 cells for 21 generations.The stable deletion of TK,gE,and gI genes was confirmed by PCR,while stable insertion of 2Cap,3Cap,and IL-4 genes was confirmed by PCR and immunofluorescence.One-step growth curves showed that rPRV-2Cap/3Cap,rPRV-2Cap/3Cap/IL4 and parental strain PRV XJ had similar growth kinetics.However,at the same time point,the titer of rPRV-2Cap/3Cap/IL4 was the lowest,followed by rPRV-2Cap/3Cap.The plaque size of rPRV-2Cap/3Cap,rPRV-2Cap/3Cap/IL4 was a significant difference in size compared to the parental strain PRV XJ.3.Evaluation of immune effect of two recombinant virus strainsAfter intramuscular immunization of B ALB/c mice with rPRV-2Cap/3Cap and rPRV2Cap/3Cap/IL4,the mice were observed continuously for 15 days to record their survival and evaluate the safety of the recombinant viruses for the mice.The results of the immunological test showed that PRV-gB-Abs,PCV2-Cap-Abs and PCV3-Cap-Abs could be detected 21 days after the first immunization,and the antibody levels gradually increased.At 35 days,the neutralizing antibody titers of PCV2 were 1:18.59 and 1:22.39,respectively.Boost immunization was given after 14 days.Serum samples were collected from mice for specific antibody detection.No PRV-gE-Abs were detected in any mice,but PRV-gB-Abs,PCV2-Cap-Abs and PCV3-Cap-Abs were detectable 21 days after the initial immunization,and antibody levels gradually increased.Serum samples were collected 14 and 28 days after the initial immunization to detect secretion of IL-6 and IFN-y,and splenocyte proliferation experiments and T lymphocyte subpopulation analysis were performed.Results showed that the expression levels of IFN-γ and IL-6 in the immunized recombinant virus group were significantly upregulated compared with the DMEM group,and lymphocyte counts were significantly increased after stimulation with the S protein,with the percentages of CD3+CD4+and CD3+CD8+T cells significantly increased.Immunohistochemical analysis of the spleen showed that rPRV-2Cap/3Cap and rPRV2Cap/3Cap/IL4 could colonize important immune organs.Six weeks after the initial immunization(Day 42),all groups of mice were challenged with a dose of 10 X LD50 of PRV.The control group began to die on the second day after inoculation with PRV XJ and all died by the fourth day,while the immunized recombinant virus group showed 100%protection against PRV.In addition,all groups of mice were challenged with a dose of 105.0 TCID50/150uL of PCV2.The tissue viral load of the immunized group peaked in the second week and gradually decreased thereafter,while the tissue viral load of the control group peaked in the third week,with no significant decrease in the fourth week,and remained higher than that of the immunized group.Moreover,the control group showed viremia,while the immunized group did not.Histopathological analysis showed that the heart,lung,spleen,and lymph nodes of the control group mice all had varying degrees of lesions,while the immunized group mice had no apparent lesions.In summary,rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 demonstrated good safety and immunogenicity in mice,and were able to induce high levels of antibodies.When mice were challenged with PRV,they showed 100%protection,and when challenged with PCV2,the virus load was reduced.Furthermore,the recombinant virus with IL-4 as an adjuvant showed superior performance in most indicators compared to the group without IL-4.Therefore,rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 have the potential to simultaneously prevent porcine circovirus type 2,type 3,and pseudorabies virus as recombinant vaccines,and IL-4 as a vaccine adjuvant can effectively enhance the immune efficacy of the vaccine. |
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