Caracterización de la infección neurológica por poliomavirus BK

Abstract

Human polyomavirus BK (BKPyV) is the aetiological agent of polyomavirus-associated nephropathy (PVAN) and of hemorrhagic cystitis (HC) while human polyomavirus JC (JCPyV) is associated with progressive multifocal leukoencephalopathy (PML). However, JCPyV can give rise to PVAN and HC and BKPyV has been detected in cerebrospinal fluid samples (CSF) from patients with Central Nervous System (CNS) disease, including PML. During the period 1998– 2011, 2,406 CSF samples from patients with suspected JCPyV infection were tested for the presence of BKPyV, JCPyV, and SV40 large T antigen DNA by a multiplex PCR assay in the National Centre of Microbiology. Twenty neurological patients with at least one BKPyV DNApositive CSF specimen were identified. Firstly, an internally-controlled multiplex real-time PCR was developed and showed to be suitable for the diagnosis of polyomavirus BK and JC infection, providing a sensitive, reproducible and specific quantification of the viral load of both viruses in samples of patients with associated pathologies. The present method was used to quantify BKPyV in the CSF of neurological patients for the first time, showing a median of 9 x 103 copies/ml (range 6 x102- 2x 106 copies/ml) and no differences were observed with JCPyV viral load in PML patients. Secondly, BKPyV non coding control region (NCCR) sequences from the 20 neurological patients were determined and compared to NCCR sequences from BKPyV variants present in renal transplant recipients, bone marrow transplant recipients and healthy pregnant women. The archetypal conformation of BK polyomavirus regulatory region was predominant in the neurological patients and in other groups of patients and controls and, therefore, rearrangements are not sufficient per se to to induce neurotropism. Five out of six early and four out of six late rearranged promoters from BK polyomavirus neurological strains showed significantly higher activity than the corresponding archetypal promoter in transfection studies with Vero cells. Thirdly, the entire VP1 protein sequence of 14 of the 20 neurological BKPyV isolates was determined and compared to VP1 sequences from strains circulating in patients with other pathological conditions such as renal transplant recipients and bone marrow transplant recipients and from healthy pregnant women. Our results revealed that VP1 reference sequence was the most common in the CSF of the neurological patients and that subtype I was predominant in all patients groups. This suggests that VP1 polymorphism is not strictly necessary or sufficient for BKPyVassociated pathology and that specific strains are not related to a specific clinical manifestation. However, a significant accumulation of amino acid substitutions in the BC loop of VP1, while a decreased tendency for EF loop mutations was found. Molecular modelling calculations indicated that the VP1 mutants may have altered host cell receptor binding properties.