BIOLOGICAL, MOLECULAR AND IMMUNOCYTOCHEMICAL CHARACTERIZATION OF CELL CULTURE ADAPTED HUMAN ROTAVIRUS STRAINS DETECTED IN THE CITY OF PONTA GROSSA, PARANA, BRAZIL

Abstract

Rotaviruses (RV) are the single most important cause of severe diarrheal illness in infants and young children in both developed and developing countries worldwide. They affect mainly children less than 24 months of age. Presently, we adapted human rotavirus strains from field materials in cell culture, as well as, studied their properties by the analyses of the cytopathic effect, immunofluorescence features, plaque properties and virus genome stability. The study was carried out with 53 rotavirus-positive fecal specimens, collected in the city of Ponta Grossa, Parana, before the introduction of vaccination program in Brazil. The samples were submitted to at least 3 blind passages in MA-104 cell culture before discarded as non-cultivable. The adaptation process was monitored by the development of the cytopathic effect, immunofluorescence assay , plaque assay and genome stability by reverse transcriptase-polymerase chain reactions amplification. Fourteen human rotavirus strains were adapted to grow in cell culture. The isolation was demonstrated by the development of typical cytopathic effect. Immunofluorescence assay revealed granular inclusions with specific fluorescence mainly at the perinuclear area of the infected cells. Plaque assay allowed the demonstration of plaques with varying diameter depending upon the strains. Reverse transcriptase-polymerase chain reactions resulted in amplification and genotyping, demonstrating the stability of the genome through cell culture adaptation. DOI: http://dx.doi.org/10.17525/vrr.v19i1.97