RESUMO
Human bocaviruses (HBoV) are non-enveloped, single-stranded DNA viruses, classified into the genus Bocavirus in the family Parvoviridae. Self-assembled virus-like particles (VLPs) composed of the major capsid protein VP2 of HBoV1-4 and mosaic VLPs composed of both VP2 and VP1 capsid proteins of HBoV1 were generated in yeast Saccharomyces cerevisiae and used to detect HBoV-specific IgG in human serum. Recombinant HBoV VLPs were similar to native HBoV particles in size and morphology. The prevalence of HBoV infection in a group of Lithuanian patients with clinical symptoms of respiratory tract infection was studied using purified yeast-generated VLPs as antigens in a competitive enzyme immunoassay (EIA). After depletion of cross-reactive antibodies, the seroprevalence of HBoV1 was 44.2 % and the seroprevalence of HBoV2-4 was 35.7 %. Mosaic VLPs consisting of HBoV1 VP1 and VP2 proteins showed a stronger reactivity with HBoV1 IgG-positive human serum specimens, and two equivocal serum specimens were reinterpreted as positive. Thus, mosaic VLPs offer a more sensitive tool for HBoV1 serology than currently available serodiagnostics tests based on VP2 VLPs. In conclusion, yeast S. cerevisiae represents an efficient expression system for generating recombinant HBoV1-4 VLPs of diagnostic relevance.
Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Bocavirus Humano/isolamento & purificação , Infecções por Parvoviridae/diagnóstico , Adolescente , Adulto , Idoso , Animais , Proteínas do Capsídeo/imunologia , Criança , Pré-Escolar , Clonagem Molecular , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Humanos , Imunoglobulina G/sangue , Lactente , Lituânia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Infecções por Parvoviridae/sangue , Infecções por Parvoviridae/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Manejo de Espécimes , Adulto JovemRESUMO
Human parvovirus 4 (PARV4) is a novel tetraparvovirus that was isolated from intravenous drug users in 2005. Recombinant PARV4 capsid protein VP2 can form stable virus-like particles (VLPs) in yeast. These VLPs could act as antigen carriers during vaccine development. Therefore, the information about PARV4 VP2 VLP antigenic sites could advance further research in this area. In this work, human parvovirus 4 VLPs obtained from yeast were used to generate monoclonal antibodies (mAbs) in mice. Epitope mapping of the obtained mAbs showed at least three distinct antigenic sites of the VP2 protein. On top of that, molecular cloning was used to replace PARV4 VP2 antigenic sites with heterologous peptides. The chimeric PARV4 VLPs bearing polyhistidine inserts obtained from yeast were observed using electron microscopy while polyhistidine-specific antibodies detected heterologous peptides of the chimeric VP2 proteins.