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BackgroundThe replication of BK virus (BKV) and JC virus (JCV) is linked to polyomavirus-associated nephropathy, hemorrhagic cystitis, and multifocal leukoencephalopathy in immunodeficient patients, but the behavior of these viruses in immunocompetent individuals has hardly been characterized MethodsWe used EIA to study samples obtained from 400 healthy blood donors aged 20–59 years for BKV- and JCV-specific antibodies against virus-like particles. We also studied BKV and JCV loads in plasma and urine among these individuals by use of real-time polymerase chain reaction ResultsIgG seroprevalence was 82% (328 of 400 donors) for BKV and 58% (231 of 400) for JCV. As age increased (age groups were divided by decade), the seroprevalence of BKV decreased from 87% (87 of 100) in the youngest group (aged 20–29 years) to 71% (71 of 100) in the oldest group (aged 50–59 years) (P=.006), whereas the seroprevalence of JCV increased from 50% (50 of 100) in the youngest group to 68% (68 of 100) in the oldest group (P=.06). Asymptomatic urinary shedding of BKV and JCV was observed in 28 (7%) and 75 (19%) of 400 subjects, respectively, with median viral loads of 3.51 and 4.64 log copies/mL, respectively (P<.001). Unlike urinary BKV loads, urinary JCV loads were positively correlated with IgG levels. The shedding of JCV was more commonly observed among individuals who were seropositive only for JCV, compared with individuals who were seropositive for both BKV and JCV, suggesting limited cross-protection from BKV immunity. Noncoding control regions were of archetype architecture in all cases, except for 1 rearranged JCV variant. Neither BKV nor JCV DNA was detected in plasma ConclusionsOur study provides important data about polyomavirus infection and replication in healthy, immunocompetent individuals. These data indicate significant differences between BKV and JCV with respect to virus-host interaction and epidemiology

Despite the high prevalence of BK polyomavirus (BKPyV) and the associated risk for BKPyV-associated nephropathy (BKPyVAN) in kidney transplant (KTX) recipients, many details on viral processes such as replication, maturation, assembly and virion release from host cells have not been fully elucidated. VP1 is a polyomavirus-specific protein that is expressed in the late phase of its replicative cycle with important functions in virion assembly and infectious particle release. This study investigated the localization and time-dependent changes in the distribution of VP1-positive viral particles and their association within the spectrum of differing cell morphologies that are observed in the urine of KTX patients upon active BKPyV infection. We found highly differing recognition patterns of two anti-VP1 antibodies with respect to intracellular and extracellular VP1 localization, pointing towards independent binding sites that were seemingly associated with differing stages of virion maturation. Cells originating from single clones were stably cultured out of the urine sediment of KTX recipients with suspected BKPyVAN. The cell morphology, polyploidy, virus replication and protein production were investigated by confocal microscopy using both a monoclonal (mAb 4942) and a polyclonal rabbit anti-VP1-specific antibody (RantiVP1 Ab). Immunoblotting was performed to investigate changes in the VP1 protein. Both antibodies visualized VP1 and the mAb 4942 recognized VP1 in cytoplasmic vesicles exhibiting idiomorphic sizes when released from the cells. In contrast, the polyclonal antibody detected VP1 within the nucleus and in cytoplasm in colocalization with the endoplasmic reticulum marker CNX. At the nuclear rim, VP1 was recognized by both antibodies. Immunoblotting revealed two smaller versions of VP1 in urinary decoy cell extracts, potentially from different translation start sites as evaluated by in silico analysis. Oxford Nanopore sequencing showed integration of BKPyV DNA in chromosomes 3, 4 and 7 in one of the five tested primary cell lines which produced high viral copies throughout four passages before transcending into senescence. The different staining with two VP1-specific antibodies emphasizes the modification of VP1 during the process of virus maturation and cellular exit. The integration of BKPyV into the human genome leads to high virus production; however, this alone does not transform the cell line into a permanently cycling and indefinitely replicating one.

BK and JC Polyomaviruses (BKPyV and JCPyV) are ubiquitous human pathogens capable of establishing lifelong, asymptomatic persistence in the majority of the global population. While decades of research have focused on their lytic replication cycles and the development of severe diseases, such as polyomavirus-associated nephropathy (PVAN) caused by BKPyV and progressive multifocal leukoencephalopathy (PML) caused by JCPyV, their primary evolutionary strategy is one of persistence rather than pathogenesis. This review shifts the perspective from a replication-centric framework towards an evolutionary persistence model, detailing the multi-layered host and viral determinants that maintain the homeostatic balance. At the cellular level, viral genomes are restricted by chromatinization into minichromosomes and host S-phase licensing. These constraints are reinforced by innate immune sensing and adaptive T-cell and antibody responses that curtail systemic dissemination while permitting periodic, low-level urinary shedding, which is essential for horizontal transmission. In addition to these host barriers, the viruses utilize intrinsic regulatory mechanisms to prevent excessive replication and immune detection, including the stable archetype non-coding control region (NCCR), viral microRNAs that downregulate early gene expression, and the small t antigen (STAg). Finally, we address unresolved questions regarding the full spectrum of cellular reservoirs, the molecular triggers of reactivation, and the ecological factors shaping their transmission routes. Understanding these maintenance mechanisms is crucial for refining clinical interventions and managing the rare, devastating transitions from silent persistence to lytic disease.

Background In kidney transplant recipients (KTR), BK polyomavirus-associated nephropathy (BKPyVAN) is a major cause of graft loss. To facilitate the clearance of BKPyV-DNAemia, reduction of immunosuppression is currently the treatment of choice but may increase the risk of graft rejection. Methods This international CERTAIN study was designed to determine the risk of alloimmune response and graft dysfunction associated with immunosuppression reduction for BKPyV treatment in 195 pediatric KTR. Results BKPyV-DNAemia was associated with a more than twofold increased risk of late T cell-mediated rejection (TCMR) (HR 2.22, p  = 0.024), of de novo donor-specific HLA antibodies (dnDSA) and/or antibody-mediated rejection (ABMR) (HR 2.64, p  = 0.002), and of graft function deterioration (HR 2.73, p  = 0.001). Additional independent risk factors for dnDSA/ABMR development were a higher HLA mismatch (HR 2.72, p  = 0.006) and re-transplantation (HR 6.40, p  = 0.000). Other independent predictors of graft function deterioration were TCMR (HR 3.98, p  = 0.003), higher donor age (HR 1.03, p  = 0.020), and re-transplantation (HR 3.56, p  = 0.013). Conclusions These data indicate that reduction of immunosuppression for BKPyV-DNAemia management is associated with increased alloimmune response in pediatric KTR. Therefore, regular dnDSA screening and close monitoring of graft function in case of BKPyV-DNAemia followed by subsequent reduction of immunosuppressive therapy are recommended. Graphical sbstract A higher resolution version of the Graphical abstract is available as Supplementary information

BK polyomavirus (BKPyV) is a major cause of kidney transplant failure, for which there are no antivirals. The current model is that BKPyV expresses TAg (large T antigen) early during infection, promoting cells to enter S phase where the viral DNA can access the host replication machinery. Here, we performed a single-cell analysis of viral TAg expression throughout the cell cycle to reveal that robust TAg expression required replication of the host DNA first. By using inhibitors that only affect host and not viral replication, we show that both TAg expression and viral production rely on an initial S phase. BKPyV is known to promote cellular re-replication, where the cell re-enters S phase from G2 phase (without passing through mitosis or G1 phase) to prolong S phase for viral replication. Thus, BKPyV infection results in cells with greater than 4N DNA content. We found that these subsequent rounds of replication of the host DNA relied on canonical host cell cycle machinery and regulators despite BKPyV infection. Together, these findings suggest a model for polyomavirus replication, where robust viral TAg expression depends on an initial host S phase and that BKPyV primarily replicates during host re-replication. Having a better understanding of the molecular events that are required for BKPyV production will help identify effective therapeutic targets against BKPyV.

Polyomaviruses have the potential to cause significant morbidity not only in transplant medicine, but also in other forms of disease or variants of immunosuppression. In kidney transplant recipients or recipients of human stem cell transplants, the BK-Virus is the major proponent of manifestations such as BKPyV-associated nephropathy or hemorrhagic cystitis. As no polyomavirus-specific drug with proven in vivo effects has been developed so far, methods to screen for such drugs are important. This work describes the establishment of a virus-secreting cell line. By infecting a pre-established monkey kidney cell line (COS-1) with a non-rearranged human BK polyomavirus isolated from a kidney transplant patient suffering from BKPyV-associated nephropathy, a continuously replicating cell type with consistent virus secretion could be established and was termed COSSA. Measurements of BKPyV replication, virion production, and secretion were performed both intracellularly and in the cell supernatant. Viral proteins such as VP1 and LTAg were accurately tracked by confocal microscopy, as well as by immunoblot and qPCR. An intracellular flow cytometry (FACS) assay detecting VP1 protein was established and revealed an expanded range of positive intracellular signals. The viruses produced proved to be infectious in human tubular epithelial cell lines. Long-range sequencing of the COSSA genome using Oxford Nanopore Technology revealed a total of five distinct BKPyV integration events. One integration of a partial BKPyV genome was located upstream of the epidermal growth factor receptor gene. The second and third, both truncated forms of integration, were close to histocompatibility gene locuses, while the fourth was characterized by a ninefold and the fifth by a fourfold tandem repeat of the BKPyV genome. From both of the repeat forms, virus replicates were derived showing deletions/duplications on early and late genes and inversions within the non-coding control region (NCCR). This pattern of repetitive viral genome integration is a potential key driver of enhanced viral replication and increased virion assembly, ultimately supporting efficient virus egress. Quantitative PCR analysis confirmed the release of approximately 108/mL viral units per 48 h from 2 × 105 COSSA cells into the culture supernatant. Notably, the NCCR region of the most frequent copies of circular virus and the integrated tetrameric tandem repeat exhibited a rearranged configuration, which may contribute to the observed high replication dynamics. The establishment of a consistent methodology to generate and secrete BKPyV from a cell line is expected to significantly facilitate antiviral drug development.

ObjectiveThis study explored the differences in intrapatient variability of tacrolimus between prolonged-release and immediate-release formulations and evaluated the association between tacrolimus intrapatient variability and reactivation of BK virus and cytomegalovirus in kidney transplant recipients.MethodsThis retrospective observational study included 270 kidney transplant recipients receiving either prolonged-release tacrolimus or immediate-release tacrolimus. Receiver operating characteristic curve analysis identified tacrolimus intrapatient variability cutoff values associated with viral reactivation. Logistic regression analyses identified predictors of BK virus and cytomegalovirus reactivation.ResultsThe prolonged-release tacrolimus group had significantly lower tacrolimus intrapatient variability than the immediate-release tacrolimus group (p < 0.001). No significant differences were observed in BK virus and cytomegalovirus reactivation rates. Receiver operating characteristic curve analysis identified tacrolimus intrapatient variability cutoffs of 0.268 for BK virus and 0.261 for cytomegalovirus. High tacrolimus intrapatient variability was significantly associated with increased BK virus and cytomegalovirus reactivation. Logistic regression showed that high tacrolimus intrapatient variability was significantly associated with BK virus and cytomegalovirus reactivation. Multivariate analysis confirmed an independent association between high tacrolimus intrapatient variability and cytomegalovirus reactivation.ConclusionsTacrolimus intrapatient variability may predict reactivation of latent viral infection after kidney transplantation. Although viral reactivation rates were similar between tacrolimus formulations, prolonged-release tacrolimus showed lower tacrolimus intrapatient variability levels, suggesting that fluctuations in tacrolimus exposure might increase the risk of viral reactivation.

BK polyomavirus (BKPyV) is a ubiquitous human virus that establishes a persistent infection in renal tubular epithelial cells and mainly causes disease in kidney transplant recipients. The closely related simian polyomavirus SV40 is known to cause cytoplasmic vacuolization in simian kidney cells, possibly increasing progeny release and cell death. This study aimed to determine whether BKPyV causes cytoplasmic vacuolization in primary human renal proximal tubule epithelial cells (RPTECs) and to investigate its potential role in the replication cycle. Using a large infectious dose (MOI 100–1000), a fraction of RPTECs (10–72%) showed early-wave vacuolization from 3 hours post-infection (hpi), which was mainly reversed by 36 hpi. Independent of the infectious dose, late-wave vacuolization occurred around the timepoint of progeny release. BKPyV receptor binding and internalization were required, as neuraminidase pretreatment and preincubation or treatment with a BKPyV-specific neutralizing antibody prevented early or late-occurring vacuolization. Microscopy revealed that the vacuoles were enlarged acidic endo-/lysosomal structures (dextran, EEA1, Rab5, Rab7, LAMP1, and/or Lysoview positive) that contained membrane-bound BKPyV. Time-lapse microscopy and quantitative PCR revealed that cell death and progeny release preceded late-wave vacuolization, mainly affecting cells directly neighboring the lysed cells. Thus, vacuolization had little impact on cell death or progeny release. Addition of the V-ATPase inhibitor Bafilomycin A1 at 0 hpi blocked vacuolization and BKPyV replication, but addition at 2 hpi only blocked vacuolization, suggesting that continuous endosomal acidification and maturation is needed for vacuole formation, but not for BKPyV replication. Our study shows that a massive uptake of BKPyV in RPTECs induces transient enlargement of endo-/lysosomes and is an early event in the viral replication cycle. Vacuolization gives no clear benefit for BKPyV and is possibly the result of a transiently overloaded endocytic pathway. Focal vacuolization around lysed cells suggests that the spread of BKPyV is preferably local.

Viral microRNAs (miRNAs) play an important role during infection by posttranscriptionally regulating both host and viral gene expression. However, the function of many viral miRNAs remains poorly understood. In this study, we investigated the role of the BK polyomavirus (BKPyV) miRNA in regulating virus replication. The function of the polyomavirus miRNA was investigated in archetype BKPyV, which is the transmissible form of the virus and thought to establish a persistent infection in the host urinary tract. In agreement with previous studies, we show that the BKPyV miRNA targets early mRNAs. Importantly, we show that the miRNA plays a significant role in limiting archetype BKPyV replication in a natural host cell model of infection. This regulation is accomplished through the balance of regulatory elements located within the noncoding control region that control early gene expression and miRNA expression before genome replication. We therefore provide evidence for a unique function of the polyomavirus miRNA that may have important implications for the mechanism of viral persistence.

BK polyomavirus (BKPyV) is a human pathogen that causes severe disease in immunocompromised individuals. Although discovered in the 1970s, important gaps in our understanding of BKPyV biology persist. Key unresolved areas include the precise molecular mechanisms governing viral latency and reactivation, the specific host and viral factors determining the virus tropism towards the urinary tract, and the intricate virus-host interactions that drive clinical pathogenesis. These unresolved biological questions have stalled the development of targeted therapeutics; as a result, no specific antiviral therapy is currently available for BKPyV-related diseases. In this review, we examine findings from both experimental models and clinical samples that investigate how BKPyV remodels host organelles and the molecular pathways underlying these alterations. We focus on BKPyV-driven changes in cellular membranes, including endoplasmic reticulum remodeling, mitochondrial disruption, the formation of endoplasmic reticulum-derived tubuloreticular structures, vacuoles, and autophagosomes, as well as the accumulation of lipid droplets. Collectively, these organelle-specific modifications highlight membrane remodeling as a central feature of BKPyV replication and pathogenesis. Addressing the key knowledge gaps in the molecular basis of virus-induced membrane remodeling will be critical for guiding the development of effective antiviral strategies.