Development Of An Indirect ELISA Method For Porcine Bocavirus And Cytopathologic Effects Of PBoV/PCV2 Co-infection

Porcine bocavirus(PBoV)is a single-stranded DNA virus firstly identified in the lymph node of swine in 2009.It has been reported worldwide in postweaning multisystemic wasting syndrome(PMWS),diarrheic,respiratory or asymptomatic swine.PBoV is presented at a high prevalence in either health or diseased swine herds around the world.Its pathogenicity remains uncertain,presumably with a weak pathogenicity and playing a pathogenic role with the help of other viruses.In field PBoV frequently co-infects with other viruses,especially Porcine Circovirus type 2(PCV2),suggesting that PCV2 may play an important role in the pathogenic process of PBoV.In this study,an indirect ELISA method for detection of PBoV antibody was developed based on the recombinant truncated PBo V VP1 protein.By detecting the expression of inflammatory factors in porcine intestinal epithelial cell IPEC-J2 infected by PBoV alone or with PCV2,we preliminary discussed the pathogenicity of PBoV,and the synergic role of PCV2 in PBoV infection.The main results were described as following: 1.Development of an indirect ELISA method for detecting PBoVantibodyPart of VP1 gene of PBoV strain NP08 was cloned and prokaryotic expressed.Then an indirect ELISA method was developed to detect antibody against PBoV with the purified recombinated VP1 protein as the coating antigen.The method had an coincidence rate of 81.25% with Western blot detection.The intra-assay and inter-assay coefficients of variation were lower than 10%.The method was used to detect 832 serum samples collected from eleven pig farms and one pig abattoir.The results showed that the positive rate of PBoV antibody was 25.6%,including 32.65%(207/634)of positive rate in diarrheic pigs,and 3.03%(6/198)in healthy pig.2.Cytopathogenic study of PBo VPorcine kidney cells PK-15 were infected with PBoV or PCV2 alone,and co-infected with PBoV and PCV2.There was no visible pathologic change in the cells of groups Mock and PCV2.Evident cytopathic effects(CPE)such as swelling,stacking and defulvium were found in groups PBoV infection and co-infection.Moreover,it was worse in group co-infection than in group PBoV infection.However,PCV2 was incapable of promoting the proliferation of PBoV in PK-15 cells detected by using real-time PCR.Then IPEC-J2 cells were infected with PBoV or PCV2 alone,and co-infected with PBoV and PCV2.Cell defulvium was observed in all infection groups detected by HE stain.The CPE occurred mainly in the cytoplasm,with phenomena of degradation,rarefaction and cavitation.At 24 h post-inbfection,the apoptosis percentages in the PBoVand PCV2-infected cells were 6.70% and 6.29%,respectively,while it was obviously higher(10.8%)in the co-infected cells.Detecting by real-time PCR,Both PBoV and PCV2 could not promote the proliferation of the other virus co-infecting IPEC-J2 cells regardless of the order of infection.Compared to the single infection group,the expression of such inflammatory factors as IL-6、IL-8、IL-1β、TNF-α in the co-infection group was extremely significantly higher at the transcriptional and translational level(P<0.001).The results of TEER showed that the value of trans epithellal electric resistance(TEER)in all the infection groups rapidly dropped and then ascended wiin 1h post infection It dropped again at 12 h post infection.Finally it came to stabilization during 12h-84 h.Further study showed that three tight junction proteins,OCLN、CLDN1、ZO-1,were significantly down-regulated in all the infection groups than the mock group(P<0.01),which mainly occurred in the early stage of viral infection.To sum up,our research firstly discussed the effect of PCV2 on the proliferation and pathogenicity of PBoV at the cell level,and found that the CPE was more serious in the co-infection group than the single infection groups.Although PCV2 was unable to promote the proliferation of PBoV,it could spur the copious expression of inflammatory factors and the damage of cell barrier in the infected cells.The study provides an evidence for the further research of the mechanism of infection and pathogenicity of PBoV.