Detection Of Bocaviruses Infection And Research On Original Antigenic Sin Of Human Bocaviruses

Bocaviruses were novel DNA viruses which were discovered from human beings, pigs, cattle, dogs and apes, and suffered from many kind of mammals in recent years. Human bocaviruses(HBoV) is firstly discovered in 2005 as new bocaviruses. HBoV have found the existence in chimpanzees, gorillas and other primates; and it shared extremely high homology with Gorillas bocavirus. Moreover, studies about HBoV immunological features will help us to have better understanding about other emerging animal bocaviruses.HBoV1 was reported widespread, and may cause acute respiratory disease in young children. Another three human bocaviruses(HBoV2-4) infection often occur in the gastrointestinal tract, were likely related to diarrhea or gastroenteritis in infants and children. It was reported that the presence of human bocaviruses were frequently detected in respiratory samples, serum samples, and also stool samples. The pathogenic mechanism of human bocaviruses is unclear. The tendency of the humoral immune response to reutilize clones activated during primary exposure to a pathogen after exposure to an antigenically similar, yet distinct version of the germ, resulting in anti-viral Abs that are of a lower affinity and less efficient neutralizers of the secondary infectious entity. A phenomenon describing such immunologic behavior among related viruses has been known since the 1950 s and termed “original antigenic sin”(OAS).In this study, three different detection methods were used for detecting HBoV DNA in nasopharyngeal aspirates- qPCR, RT-PCR and MariPOC antigen diagnosis system. In those 269 NPA samples, the positivity of HBoV1 DNA in q PCR is 8.1%(22/269) and DNA copies were between 5.13E+01 to 7.23E+09 copies/mL. The positivity in mRNA was 6.16%(9/146) and the positivity in MariPOC antigen detection system was 6.8%(10/146). The minimal limit of those two latter detection methods is about 107 copies/mL. The preliminary speculation is that HBoV1 DNA should more than 107 copies/ mL in the respiratory tract samples during acute viral infection.To study these interactions of HBoV infections, we examined ~2000 sera collected at 3-6 month intervals from over 100 children. The highest prevalence around four HBoV DNA was HBoV1-24%; the prevalence of HBoV2-4 DNA was 6%, 1% and 1%, respectively. By the HBoV1-3 IgG ELISA, the positivity of HBoV1-3 was 86.2%, 53.2% and 10.1%, respectively. By the HBoV1-3 IgM ELISA, HBoV1 IgM positivity was 32%, HBoV2 was 10%, and none of the samples was HBoV3-IgM positive. In our children serology study, among the children with more than one type of HBV infection, after secondary HBV infection, some individuals produce the antibodies against primary HBoV infection rather than secondary HBoV. Based on those data, we believed the presence of OAS among HBoV 1-4 infection in human.To characterize this putative OAS phenomenon in a more controlled setting, we planed to construct HBoV4 VP2 VLPs and expressed large quantity of the HBoV1-4 VP2 VLPs by baculovirus-insect cell expression system; while we optimized the detection methods which will be used to test the effect of the original antigen sin. First of all, baculovirus expression vector pAcUW51-HBoV4-VP2 was successfully constructed. The recombinant bacmids were transfected into Sf9 insect cells and harvested after 3 days post-transfection. The recombinant bacmids were further propagated and then to express the recombinant proteins in High5 insect cells. With SDS-PAGE, the size of expression recombinant proteins was 62 kDa. Western blot results showed specific reaction with HBoV4 positive serum, with excellent antigenicity. After cesium chloride density gradient centrifugation of the cell lysate supernatants, about 22 nm particles were presented in electron microscopy, which is similar to HBoV4. So HBoV4 VLPs were successfully constructed and further confirmed. Secondly, by the baculovirus-insect cell expression system, we expressed large quantity of HBoV1-4 VP2 VLPs, and purified proteins by cesium chloride ultracentrifugation. With SDS-PAGE, proteins were displayed with predicted size and the good purity. Using non-blocking and blocking Ig G ELISA, HBoV1-4 VP2 VLPs were proven with good antigenicity.Then, we preliminarily applied VLPs in HBoV4 IgG ELISA. The cut-off value was established as follows: OD492nm≥0.243, positive; OD492nm<0.243, negative. The coincidence rate of the 10 existing HBoV4 positive samples was 100%. After blocking with HBoV1-3 antigens, the prevalence of HBoV4 antibodies in192 children sera was 3.6%(7/192). At the final, we modified the existing HBoV1-4 indirect IgG ELISA for the rabbit sera. After improvement, the optimal dilution of rabbit serum was 1: 1000; the optimal concentration of blocking antigen was 30 mg / mL and the optimal concentration of swine anti-rabbit IgG / HRP was 1: 2000.With 10 rabbit pairs, inoculated by HBoV1-4 VLPs and human parvovirus B19 VLPs in various combinations. The secondary HBoV or B19 antigen was injected 60 days after the first HBo V or B19 antigen, and serum was regularly collected until day 120 post-inoculation. By analyzing the sera with HBoV1-4 IgG EIAs with and without blocking away heterologous HBoV antibodies, all rabbits showed a clear antibody response. However, there were no significant IgG boosts after the second inoculation in either the homologous HBoV1 or HBoV2-inoculated rabbits. B19 V with HBoV1 does not affect the original antigen sin. Among heterologous HBoV-inoculated combinations, half of individuals showed OAS of different degrees, similarly to our results of human sera, which is similar to the results of our previous studies in human serum. Through third-party HBoV IgG ELISA, the level of cross-reactive antibodies was reflected. For secondary antigen as HBoV1, there is 66% occurrence of possible original antigenic sin. With secondary antigen as HBoV2, it was less likely occurred original antigenic sin.HBoVs are the first parvoviruses to show the OAS phenomenon. Our findings provide new information on HBoV1-4 immunity and emphasize the complexity of human bocavirus diagnosis, and also provided the theoretical basis for development of HBoV serology diagnosis and vaccines in future.