Studies On Serologically Differential Diagnosis Between Porcine Circovirus Type 1 And 2

Porcine circovirus (PCV) has two serotypes. One is porcine circovirus type 1 (PCV1) which is nonpathogenic. Another is porcine circovirus type 2 (PCV2) which can induce immunosuppression and is considered to be an important emerging pathogen associated with a number of di?erent syndromes and diseases in pigs such as postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), porcine respiratory disease complex, reproductive failure, and so on. There are cross-reactivity due to 85% homology in ORF1 between PCV1 and PCV2. In addition, PCV1 and PCV2 as a mixture infection emerge in the field condition. It is important to develop differential diagnosis methods for PCVs. An immunoperoxidase monolayer assay (IPMA) and a blocking ELISA based on a monoclonal antibody (McAb) for neutralization antibody detection of PCV2 were developed and validated in this study. The two assays offer some convenient tools for epidemiology survey and vaccine evaluation. McAbs also can be used for antigen epitope analysis of PCVs.An IPMA was developed for antibody detection of PCV1 in the study. The reaction, specificity, sensitivity, and repeatability of IPMA were optimized and evaluated, and compared with ELISA using baculovirus-expressed PCV1 recombinant capsid protein as antigen. In results, different dilution of PCV1 and PCV2 positive reference sera were assayed by the IPMA, and there was a low level antibody cross-reaction between two viruses could be eliminated by serum dilution more than 1:100. There was no cross-reaction with reference sera against other porcine viruses. In comparison, 92.6% agreement rate was obtained between IPMA and an ELISA using baculovirus-expressed PCV1 recombinant capsid protein as antigen. Total 257 serum samples from Heilongjiang, Jilin, Hebei, Shanghai, Liaoning provinces were detected using PCV1-IPMA and PCV2-IPMA. The positive rate was 19.1% and 80.5% for PCV1 and PCV2, respectively. About 95.9% of PCV1-positive samples belong to PCV2-pisitive either. These data demonstrates that these pig herds has contaminated with PCV1. PCV1 and PCV2 as a mixture infection emerge under the clinic condition. PCV1-IPMA offers a convenient tool for epidemiology survey and evaluation of antibodies of the virus.PCV1 recombinant Cap (PCV1-rCap) protein was expressed by recombinant baculovirus (rBac/PCV1-Cap). PCV1-rCap protein expression parameters, purification and immunological activity were studied. The recombinant protein yield was about 20.7% of the total proteins after optimizing the expression. PCV1-rCap protein was purified by inclusion body purification method, the purity and density reached 85.1% and 1.123 mg/mL. The recombinant protein was identified by Western blot using the virus-specific antibodies, and had good immunoreactivity. The rabbit serum against PCV1-rCap and PCV2-rCap were produced and could be detected by IPMA. As a result, there was no cross reaction between above anti-serum. No cross reaction between the two Cap of PCV1 and PCV2 was concluded. Therefore, Cap was considered as a target protein in the studies of differential diagnosis between porcine circovirus type 1 and 2. BALB/c mice were immunized using the PCV1-rCap and PCV2-rCap prepared by baculovirus expression system. Nine McAbs, named as 2C10, 3A8, 3F7, 5D1 and 1D2, 2E8, 3A10, 5F2, 6F10 to PCV1-rCap and PCV2-rCap were prepared by lymphocyte hybridoma technique. Nine McAbs supernatant and ascites titers were 1:320～1:10240 and 1: 320000～1:5120000. The McAbs of PCV1 subclass belonged to IgG1 for 2C10 and 3A8, IgM for 3F7, IgG3 for 5D1, and all of McAbs light chains belonged toκchain. The McAbs of PCV2 subclass belonged to IgG2a for 1D2, 2E8, 5F2 and 6F10; and only IgG1 for 3A10. The McAb light chains belonged toλchain for 2E8, 3A10, 5F2 and 6F10, and onlyκchain for 1D2. Western blot analysis showed that all of McAbs to PCV1 were able to react with native PCV1-Cap. McAbs of 2E8, 3A10, 5F2 and 6F10 were able to react with native PCV2-Cap, but not for 1D2. However, the McAb of 1D2 was able react with PCV2 by IPMA and antigen capture ELISA, and it could be differentiated with PCV1 at the same condition. The McAb of 1D2 was confirmed to have the neutralizing ability to PCV2 by neutralization test. These McAbs which to the PCVs-rCap were obtained in the study would be applied in the differential diagnostic method and antigen epitope analysis of the virus.A blocking ELISA was developed for detecting neutralization antibodies against PCV2 based on a McAb to the virus, which was prepared by lymphocyte hybridoma technique. The virus antigens were captured by PCV2 specific positive sera as coating antigen and reacted with the sera antibodies and peroxidase from horseradish (HRP)-labeled with the McAb, respectively. The results were developed by detection of the HRP-labeled McAb. In comparison with IPMA, there was 96.0% coincidence between the blocking ELISA and IPMA based on detection of 353 serum samples. The sensitivity and specificity of the blocking ELISA were 97.9% and 92.4% and showed no cross-reactivity with the reference sera of other swine viruses. The results of detection PCV2 neutralization antibodies in serum by blocking ELISA and serum neutralization test(SNT) show that the two methods were significant positive correlation (r=0.9970). The blocking ELISA was more sensitive than SNT. Neutralization antibodies could be detected from the vaccinated swine sera with PCV2 inactivated vaccine after two weeks, 100% positive after four weeks. Test of 703 serum samples collected from the provinces of Heilongjiang, Jilin, Liaoning showed 73.4% positive rates. These data demonstrate that pig herds in China have been contaminated heavily with PCV2. Thus, the blocking ELISA offers a convenient tool for epidemiology survey and evaluation of neutralization antibodies of PCV2.