The human polyomavirus KI: A study on cell permissivity, sub-cellular localization of VP1 and presence in cerebrospinal fluid and urine

Abstract

KI polyomavirus (KIPyV) is a relatively newly discovered human polyomavirus originally identified in respiratory tract samples. Little is known about the route of infection or transmission, and the pathogenic properties of this virus are virtually unknown. The life cycle of KIPyV has not been studied as a permissive cell system has not been identified so far. The bona fide sites of viral replication in the human body remain unknown, but indications of a respiratory or oral route of infection led us to investigate whether the A549 lung cell line was permissive to KIPyV. In this thesis we have investigated if the A549 cell line allows KIPyV propagation and if viral protein is detectable by antibodies directed against KIPyV VP1. This was performed by transfecting with KIPyV genome and analyzing the cells for mRNA expression of LT-ag and antibody detection of VP1 by western blotting. LT-ag mRNA was successfully detected by PCR but detection of KIPyV VP1 by the use of our antibody was unsuccessful. The sub-cellular localization of KIPyV VP1 protein has been investigated by confocal microscopy using EGFP-KI VP1 fusion protein. Both A549 and Vero cell line were studied and the EGFP signal localized differently in the two cell lines. In A549 cells the localization was mainly in the nucleus while in Vero cells cytoplasm localization was mostly observed. We have also examined cerebrospinal fluid from patients with suspicion of neurological diseases and urine specimens from immunocompromised patients with systemic lupus erythematosus for KIPyV DNA by nested PCR. We have indicated the presence of KIPyV VP1 DNA by nested PCR and sequencing of the PCR products.