BK virus (BKV) is a ubiquitous polyomavirus known to asymptomatically reside in the renal tissues of up to 90% of the human population. BK virions reactivate during periods of intense immunosuppression and can cause disease in renal transplant recipients, such as BKV-associated nephropathy (BKVAN). BKVAN can lead to loss of the transplanted renal grafts. For this reason, the study of BKV biology is of importance to the transplant community. Previous studies have shown that BKV upregulates the pro-growth mTOR pathway in host cells, thereby increasing BKV replicative efficiency. Downstream effectors of the mTOR pathway, particularly p70S6 kinase, control the basal rate of protein translation, in part through regulation of ribosomal biogenesis. It was hypothesized that viral upregulation of the mTOR pathway is beneficial for viral replication due to an increase in the number of ribosomes available to translate viral proteins. Therefore, inhibition of the mTOR pathway could reduce viral replication. This study investigated whether host cell mTOR inhibition could reduce BK viral replication in an in vitro model. We utilized the dual PI3K/mTOR inhibitor NVP BEZ-235 (Novartis Pharmaceuticals), which potently downregulates expression of both upstream (PI3K) and central (mTOR) effectors of the mTOR pathway. Immortalized renal epithelial cells were exposed to varying concentrations of BEZ-235 for a period of 48 hours, infected with BK virus for three hours, and allowed to grow for a further 48 hours. Cell populations were then assayed via quantitative PCR (qPCR), Western blotting and fluorescent immunohistochemical staining to determine the effect of BEZ-235 on BK viral replication. Western blot experiments confirmed the effectiveness of BEZ-235's inhibition of the mTOR pathway in a renal epithelial cell culture model, as well as downregulation of the mTOR pathway during BK viral infection. Western blotting for the key BK replicative protein Large T antigen showed a dose-dependent decrease in expression, with increasing concentrations of BEZ-235. Fluorescent immunohistochemical staining showed a dose-dependent decrease in expression of Large T antigen staining in host cell nuclei. qPCR results were inconclusive, in that no clear pattern in the number of BKV genomes per cell population could be observed across the range of BEZ-235 concentrations tested. While results from our study indicate that BEZ-235 can reduce BKV replication in vitro, further in vitro experimentation, including repetition of approaches already carried out as well as novel approaches, will be needed to definitively confirm inhibition of the mTOR pathway as a viable antiviral strategy.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

La néphropathie à BK virus (BKV) est l'une des complications les plus fréquentes de la transplantation rénale. La prise en charge consiste en la réduction préemptive de l'immunosuppression basée sur le suivi de la charge virale, mais cette stratégie n’est pas complètement efficace et augmente le risque de rejet. Dans un premier volet, nous avons évalué la mesure de la charge virale par PCR quantitative en temps réel, permettant de mettre en évidence des facteurs de variabilité comme le polymorphisme du BKV et de valider la technique utilisée pour le suivi de notre cohorte. L’intérêt des anticorps neutralisants (AcNs) anti-BKV en tant que marqueur prédictif de la réplication BKV a ensuite été évalué dans une cohorte de 168 transplantés rénaux. Nous avons montré i) que le virus responsable de l’infection provenait du donneur ; ii) que les AcNs jouent un rôle dans la prévention de la réactivation et le contrôle de la réplication virale et iii) qu’un seuil d’AcNs de 4 log10 permettait de stratifier le risque de réplication BKV. Ce travail ouvre la voie à un suivi personnalisé en fonction du risque de réplication BKV et à de nouvelles approches immunothérapeutiques. / BK virus (BKV)-associated nephropathy is one of the major causes of graft dysfunction and loss in kidney transplant recipients. Since no BKV-specific antiviral therapies are available, management relies on preemptive immunosuppression reduction based on viral load monitoring. However, this strategy does not fully eliminate the risk of nephropathy and can increase the risk of graft rejection. In this work, we evaluated viral load measurement by quantitative real-time PCR in an interlaboratory comparison. Variability factors such as BKV polymorphism or pre-PCR steps have been highlighted and the method used for monitoring our cohort has been validated. The role of anti-BKV neutralizing antibodies (NAbs) as a predictive marker of BKV replication has been investigated in a cohort of 168 kidney transplant recipients. We showed that i) viral infection is caused by the donor strain; ii) NAbs play an essential role in viral replication prevention and control and iii) a NAbs cutoff of 4 log10 allows to stratify BKV replication risk. This work paves the way for personalized monitoring according to BKV replication risk and for new preventive or therapeutic strategies.

Even though polyomaviruses have been intensively studied for more than 60 years, the role of minor structural proteins VP2 and VP3 in some important steps of viral life cycle has still not been fully elucidated, explicitly their role in viral genome delivery to the cell nucleus and their involvement in late phases of viral life cycle. This diploma thesis focuses on the study of minor proteins of Mouse polyomavirus (MPyV) and Human polyomavirus BK (BKV). Four rabbit polyclonal antibodies against minor proteins of polyomaviruses MPyV or BKV have been prepared within this diploma thesis. Two of these prepared antibodies target minor proteins of MPyV (α-MPyV VP2/3) or BKV virus (α-BKV VP2/3), other two prepared antibodies recognize C-terminal sequence common to minor proteins VP2 and VP3 of MPyV (α-MPyV C-termVP2/3) or BKV virus (α-BKV C-termVP2/3). In the second part of this diploma thesis we aimed to study toxicity of BKV virus minor proteins during individual production in mammalian cells. Obtained results suggest that minor proteins of BKV virus might not exhibit as high levels of cytotoxicity as minor proteins of MPyV virus. Third part of this diploma thesis is devoted to investigation of interactions of BKV and MPyV minor proteins with cellular proteins and within one another respectively....

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

Background: In the United States, increasing ethnic diversity has been apparent. However, the epidemiology and trends of BKV genotypes remain unclear. This meta-analysis was conducted with the aim to assess the prevalence of BKV genotypes among kidney transplant (KTx) recipients in the United States. Methods: A comprehensive literature review was conducted through October 2018 utilizing MEDLINE, Embase, and Cochrane Database to identify studies that reported the prevalence of BKV subtypes and/or subgroups in KTx recipients in the United States. Pooled prevalence rates were combined using random effects, generic inverse variance method. The protocol for this study is registered with PROSPERO (no. CRD42019134582). Results: A total of eight observational studies with a total of 193 samples (urine, blood, and kidney tissues) from 188 BKV-infected KTX recipients were enrolled. Overall, the pooled estimated prevalence rates of BKV subtypes were 72.2% (95% confidence of interval [CI]: 62.7-80.0%) for subtype I, 6.8% (95% CI: 2.5-16.9%) for subtype II, 8.3% (95% CI: 4.4-15.1%) for subtype III, and 16.1% (95% CI: 10.4-24.2%) for subtype IV, respectively. While metaregression analysis demonstrated a significant positive correlation between year of study and the prevalence of BKV subtype I (slopes = +0.1023, P =.01), there were no significant correlations between year of study and percentages of BKV subtype II-IV (P >.05). Among KTx recipients with BKV subtype I, the pooled estimated percentages of BKV subgroups were 22.4% (95% CI: 13.7-34.5%) for subgroup Ia, 30.6% (95% CI: 17.7-47.5%) for subgroup Ib1, 47.7% (95% CI: 35.8-59.9%) for subgroup Ib2, and 4.1% (95% CI:1.2-13.3%) for subgroup Ic, respectively. Conclusion: BKV subtype I is the most prevalent subtype among KTx recipients in the United States and its prevalence seems to increasing overtime. Subgroup Ib2 is the most common subgroup among BKV subtype I.

Polyoma BK virus is associated with graft dysfunction leading to BK viral nephropathy (BKVN) in 1-10% of kidney transplant recipients, moreover 30-80% of kidney transplant recipients experience asymptomatic reactivation of the virus that does not result in BKV associated damage of the renal allograft. The first aim of this study was to introduce monitoring of BK virus replication in the blood and urine of patients within first year after transplantation. Risk factors were evaluated and limit values for viremia and viruria for BKVN development was established. Positive BK viruria >107 copies/ml and positive BK viremia >104 copies/ml occurred in 25.8% and 5%; respectively. 3 patients out of monitoring study developed BKVN. Using ROC analysis, limit values for the development of BKVN were set at 103 copies/ml serum for BK viremia and 6.7x107 copies/ml BK viruria. The second objective was to determine the expression profile of the immune genes in kidney biopsies in three groups of patients with varying degrees of reactivation of the BK virus (without virus reactivation, with asymptomatic viruria, BKVN). 90 genes of immune response were measured by the TaqMan® low density array RT-qPCR. The analysis of biopsies from patients with non-signalling viruses led to the identification of 5 differentially...

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Polyomaviruses are small unenvelope DNA viruses, whose replication take place in cell nucleus. Despite its small genome size, these viruses can cause significant changes in the host cell, one of the most significant is cell transformation. Most studies of human pathogens from this family is the focus of clinical research, but do not provide enough information about the individual events of the life cycle of viruses. This thesis mainly aims to determining the exact time when the creation of the individual viral products and generate a timeline of events during natural infection in cells that are targets for BKV in the human body. It was found that the time course of the life cycle of BKV is very similar to those for model polyomaviruses MPyV and SV40 and in permissive cells takes about 40 - 50 hours.

Aim of this diploma thesis was to prepare two protein antigens and two monoclonal antibodies, all based on VP2 minor protein of human polyomaviruses BK virus and Merkel Cell Polyomavirus. One monoclonal antibody was being prepared against unique part of VP2 protein (N-terminal epitope, not present in VP3 protein). A cell line producing such monoclonal antibody has never been established before due to low immunogenicity of the epitope. Our approach was successful in terms of mouse immunization, however, serious problems with hybridoma line stability appeared later during the preparation process. Preparation of antibody targeted to the sequence of VP2 protein of Merkel Cell Polyomavirus was another aim of this thesis. Mouse immunization and hybridoma fusion were performed successfully. After four rounds of cloning in order to purify an established clone, nine clones were cultivated in larger scale. This cultivation probably led to diminished antibody specificity and loss of production ability in most of the hybridoma cells. One more cloning should give rise to an established clone with sufficient production. Two preparations of protein antigens were performed in two expression systems. DNA encoding C-terminally truncated protein VP2 of BK virus fused with His-tag was cloned into a vector suitable for...