| Abstract [eng] |
This PhD thesis focused on the investigation of yeast S. cerevisiae expression system for synthesis of recombinant structural proteins of novel human and economically relevant porcine parvoviruses for virus prevalence studies. In this work for the first time in yeast major structural proteins (VP2) of human parvovirus 4 (PARV4), human bocaviruses (HBoV) 1, 2, 3, and 4, as well as porcine parvovirus (PPV) were synthesised. The recombinant VP2 proteins were purified, and their ability to self-assemble into immunogenic virus-like particles was demonstrated. These antigenic VLPs were employed to estimate for the first time the prevalence of PARV4 and HBoV1-4 viruses within Lithuanian population. Using the recombinant yeast-generated parvoviral VLPs serodiagnostic assays were created and estimated, that 91.6 % of Lithuanian patients with clinically confirmed respiratory tract infections had antibodies recognising HBoV VP2 proteins. After the elimination of cross-reactive antibodies in the competition enzyme immunoassay, 44.2 % patients had HBoV1, and 35.7% of patients had HBoV2-4 specific antibodies. The serological study of PARV4 prevalence among low-risk individuals in Lithuania revealed the highest rate (9.4 %) reported in Europe to date. Taking into consideration that the patients had no records of parenteral exposure, this study supported the raising objection to the prevailing theory of parenteral PARV4 transmission mode. Detailed B-cell epitope mapping was done to confirm three main epitope locations in PARV4 VP2 protein. Yeast-generated porcine parvovirus VP2 VLPs were evaluated to be suitable for use in virus-specific antibody detection assay and to elicit the PPV-specific immune response in mice. To our best knowledge, the recombinant mosaic virus-like particles composed of major and minor structural parvoviral proteins were generated for the first time in yeast expression system. These mVLPs of HBoV1 and PARV4 were evaluated as more sensitive antigens in EIA in comparison to VP2-only VLPs. All these results show that yeast S. cerevisiae is suitable expression system to generate valuable parvoviral antigens. |
