Performance evaluation of the high-throughput quantitative Alinity m BK virus assay.

BK virus (BKV) infection or reactivation in immunocompromised individuals can lead to adverse health consequences including BKV-associated nephropathy (BKVAN) in kidney transplant patients and BKV-associated hemorrhagic cystitis (BKV-HC) in allogeneic hematopoietic stem cell transplant recipients. Monitoring BKV viral load plays an important role in post-transplant patient care. This study evaluates the performance of the Alinity m BKV Investigational Use Only (IUO) assay. The linearity of the Alinity m BKV IUO assay had a correlation coefficient of 1.000 and precision of SD ≤ 0.25 Log IU/mL for all panel members tested (2.0-7.3 Log IU/mL). Detection rate at 50 IU/mL was 100%. Clinical plasma specimens tested comparing Alinity m BKV IUO to ELITech MGB Alert BKV lab-developed test (LDT) on the Abbott m2000 platform using specimen extraction protocols for DNA or total nucleic acid (TNA) resulted in coefficient of correlation of 0.900 and 0.963, respectively, and mean bias of 0.03 and -0.54 Log IU/mL, respectively. Alinity m BKV IUO compared with Altona RealStar BKV and Roche cobas BKV assays demonstrated coefficient of correlation of 0.941 and 0.980, respectively, and mean bias of -0.47 and -0.31 Log IU/mL, respectively. Urine specimens tested on Alintiy m BKV IUO and ELITech BKV LDT using TNA specimen extraction had a coefficient of correlation of 0.917 and mean bias of 0.29 Log IU/mL. The Alinity m BKV IUO assay was performed with high precision across the dynamic range and correlated well with other available BKV assays. IMPORTANCE: BK virus (BKV) in transplant patients can lead to adverse health consequences. Viral load monitoring is important in post-transplant patient care. This study evaluates the Alinity m BKV assay with currently available assays.

Accuracy of quantitative viral secondary standards: a re-examination.

Interlaboratory agreement of viral load assays depends on the accuracy and uniformity of quantitative calibrators. Previous work demonstrated poor agreement of secondary cytomegalovirus (CMV) standards with nominal values. This study re-evaluated this issue among commercially produced secondary standards for both BK virus (BKV) and CMV, using digital polymerase chain reaction (dPCR) to compare the materials from three different manufacturers. Overall, standards showed an improved agreement compared to prior work, against nominal values in both log10 copies/mL and log10 international unit (IU)/mL, with bias from manufacturer-assigned nominal values of 0.0-0.9 log10 units (either copies or IU)/mL. Standards normalized to IU and those values assigned by dPCR rather than by real-time PCR (qPCR) showed better agreement with nominal values. The latter reinforces prior conclusions regarding the utility of using such methods for quantitative value assignment in reference materials. Quantitative standards have improved over the last several years, and the remaining bias from nominal values might be further reduced by universal implementation of dPCR methods for value assignment, normalized to IU. IMPORTANCE: Interlaboratory agreement of viral load assays depends on accuracy and uniformity of quantitative calibrators. Previous work, published in JCM several years ago, demonstrated poor agreement of secondary cytomegalovirus (CMV) standards with nominal values. This study re-evaluated this issue among commercially produced secondary standards for both BK virus (BKV) and CMV, using digital polymerase chain reaction (dPCR) to compare the materials from three different manufacturers. Overall, standards showed an improved agreement compared to prior work, against nominal values, indicating a substantial improvement in the production of accurate secondary viral standards, while supporting the need for further work in this area and for the broad adaption of international unit (IU) as a reporting standard for quantitative viral load results.

Performance of the cobas EBV and cobas BKV assays: multi-site comparison of standardized quantitation.

Guidelines recommend monitoring of Epstein-Barr virus (EBV) and BK virus (BKV) in solid organ and hematopoietic stem cell transplant patients. The majority of quantitative DNA testing for EBV and BKV employs unstandardized individual laboratory-developed testing solutions (LDTs), with implications for accuracy, reproducibility, and comparability between laboratories. The performance of the cobas EBV and cobas BKV assays was assessed across five laboratories, using the World Health Organization International Standards (WHO IS) for EBV and BKV, and the National Institute of Standards and Technology Quantitative Standard for BKV, and results were compared with the LDTs in use at the time. Methods were also compared using locally sourced clinical specimens. Variation was high when laboratories reported EBV or BKV DNA values using LDTs, where quantitative values were observed to differ by up to 1.5 log10 unit/mL between sites. Conversely, results from the cobas EBV and cobas BKV assays were accurate and reproducible across sites and on different testing days. Adjustment of LDTs using the international standards led to closer alignment between the assays; however, day-to-day reproducibility of LDTs remained high. In addition, BKV continued to show bias, indicating challenges with the commutability of the BKV International Standard. The cobas EBV and cobas BKV assays are automated, aligned to the WHO IS, and have the potential to reduce the variability in viral load testing introduced by differences in LDTs. Standardization of reporting values may eventually allow different centers to compare data to allow clinical decision thresholds to be established supporting improvements in patient management.IMPORTANCEThe application of center-specific cut-offs for clinical decisions and the variability of LDTs often hinder interpretation; thus, the findings reported here support the need for standardization in the field of post-transplant monitoring of EBV and BKV to improve patient management. Alongside the choice of assay, it is also important to consider which standard to use when deciding upon a testing methodology. This is a call to action for standardization, as treatment for EBV and BKV is driven by viral load test results, and the more accurate and comparable the test results are across institutions, the more informed and better the treatment decisions can be.

Cultured Renal Proximal Tubular Epithelial Cells Resemble a Stressed/Damaged Kidney While Supporting BK Virus Infection.

BK virus (BKV; human polyomavirus 1) infections are asymptomatic in most individuals, and the virus persists throughout life without harm. However, BKV is a threat to transplant patients and those with immunosuppressive disorders. Under these circumstances, the virus can replicate robustly in proximal tubule epithelial cells (PT). Cultured renal proximal tubule epithelial cells (RPTE) are permissive to BKV and have been used extensively to characterize different aspects of BKV infection. Recently, lines of hTERT-immortalized RPTE have become available, and preliminary studies indicate they support BKV infection as well. Our results indicate that BKV infection leads to a similar response in primary and immortalized RPTE. In addition, we examined the patterns of global gene expression of primary and immortalized RPTE and compared them with uncultured PT freshly dissociated from human kidney. As expected, PT isolated from the healthy kidney express a number of differentiation-specific genes that are associated with kidney function. However, the expression of most of these genes is absent or repressed in cultured RPTE. Rather, cultured RPTE exhibit a gene expression profile indicative of a stressed or injured kidney. Inoculation of cultured RPTE with BKV results in the suppression of many genes associated with kidney stress. In summary, this study demonstrated similar global gene expression patterns and responses to BKV infection between primary and immortalized RPTE. Moreover, results from bulk transcriptome sequencing (RNA-seq) and SCT experiments revealed distinct transcriptomic signatures representing cell injury and stress in primary RPTE in contrast to the uncultured, freshly dissociated PT from human kidney. IMPORTANCE Cultured primary human cells provide powerful tools for the study of viral infectious cycles and host virus interactions. In the case of BKV-associated nephropathy, viral replication occurs primarily in the proximal tubule epithelia in the kidney. Consequently, cultured primary and immortalized renal proximal tubule epithelial cells (RPTE) are widely used to study BKV infection. In this work, using bulk and single-cell transcriptomics, we found that primary and immortalized RPTE responded similarly to BKV infection. However, both uninfected primary and immortalized RPTE have gene expression profiles that are markedly different from healthy proximal tubule epithelia isolated directly from human kidney without culture. Cultured RPTE are in a gene expression state indicative of an injured or stressed kidney. These results raise the possibility that BKV replicates preferentially in injured or stressed kidney epithelial cells during nephropathy.

Long-read sequencing reveals complex patterns of wraparound transcription in polyomaviruses.

Polyomaviruses (PyV) are ubiquitous pathogens that can cause devastating human diseases. Due to the small size of their genomes, PyV utilize complex patterns of RNA splicing to maximize their coding capacity. Despite the importance of PyV to human disease, their transcriptome architecture is poorly characterized. Here, we compare short- and long-read RNA sequencing data from eight human and non-human PyV. We provide a detailed transcriptome atlas for BK polyomavirus (BKPyV), an important human pathogen, and the prototype PyV, simian virus 40 (SV40). We identify pervasive wraparound transcription in PyV, wherein transcription runs through the polyA site and circles the genome multiple times. Comparative analyses identify novel, conserved transcripts that increase PyV coding capacity. One of these conserved transcripts encodes superT, a T antigen containing two RB-binding LxCxE motifs. We find that superT-encoding transcripts are abundant in PyV-associated human cancers. Together, we show that comparative transcriptomic approaches can greatly expand known transcript and coding capacity in one of the simplest and most well-studied viral families.

Molecular epidemiology and risk factors associated with BK and JC polyomavirus urinary shedding after kidney allograft.

Polyomaviruses BK (BKPyV) and JC (JCPyV), belonging to the Polyomaviridae, are responsible for human pathologies. In kidney transplant recipients, BKPyV replication can lead to irreversible nephron damage whereas JCPyV replication remains asymptomatic. Concomitant replication is rare and potential competition between the infections has been described. The aim of this retrospective case-control study was to describe the molecular epidemiology and risk factors associated with BKPyV and JCPyV replication in a cohort of kidney transplant recipients. In total, 655 urine samples from 460 patients were tested for BKPyV and JCPyV DNA. Positive samples were submitted to strain genotyping. Demographic and clinical characteristics were also compared. Isolated JCPyV and BKPyV was found in 16.5% and 23.3% of patients, respectively; co-replication was rare (3.9%). BKPyV strains Ib-2, Ib-1, and IVc-2 were the most prevalent. JCPyV strains mostly belonged to genotypes 4 and 1B. During follow-up, JCPyV shedding significantly reduced the risk of BKPyV DNAuria, with an odds ratio of 0.57 (95% confidence interval: 0.35-0.99), and was associated with better prognosis than BKPyV replication, based on the estimated glomerular filtration rate. Molecular epidemiology of BKPyV and JCPyV strains in our region was similar to previous studies. This study suggests that JCPyV is benign and appears to limit damaging BKPyV replication. JCPyV DNAuria screening could thus be a useful strategy to predict BKPyV-related outcomes.

Safety and efficacy of a reduced frequency viral monitoring strategy for Epstein-Barr virus, cytomegalovirus, and BK polyomavirus post-kidney transplant: A quality assurance initiative.

BACKGROUND: There is variability in recommended viral monitoring protocols after kidney transplant. In response to increased demand for laboratory testing during the COVID-19 pandemic, the Transplant Manitoba Adult Kidney Program updated its monitoring protocols for cytomegalovirus (CMV), Epstein-Barr virus (EBV), and BK polyomavirus (BKV) to a reduced frequency. METHODS: This single-center nested case-control study evaluated 252 adult kidney transplant recipients transplanted from 2015 to 2021, with the updated protocols effective on March 19th 2020. Cases included recipients transplanted after the protocol update who developed CMV, EBV, and BKV DNAemia and were matched to controls with DNAemia transplanted prior to the protocol update. The primary outcome was the difference in maximum DNA load titers between cases and matched controls. Secondary outcomes included time to initial DNAemia detection and DNAemia clearance. Safety outcomes of tissue-invasive viral disease were described. RESULTS: There were 216 recipients transplanted preupdate and 36 recipients postupdate. There was no difference between cases and controls in maximum or first DNA load titers for EBV, CMV, or BKV. Cases experienced earlier EBV DNAemia detection (26 (IQR 8, 32) vs. 434 (IQR 96, 1184) days, p = .005). Median follow-up was significantly longer for recipients transplanted preupdate (4.3 vs. 1.3 years, p < .0001). After adjusting for follow-up time, there was no difference in DNAemia clearance or tissue-invasive viral disease. CONCLUSION: Our findings suggest that reduced frequency viral monitoring protocols may be safe and cost-effective. This quality assurance initiative should be extended to detect longer-term and tissue-invasive disease outcomes.

Molecular monitoring of viral infections in immunocompromised patients in a large university hospital in Italy: reflections after thirteen years of real-life activity.

PURPOSE: This study aimed to investigate the prevalence and viral reactivations of clinical interest in the immunocompromised patient with particular focus on hematologic and solid organ transplant recipients. METHODS: Molecular screening data of CMV, EBV, JCV and BKV from 2011 to 2023 were analyzed. This extensive time span allowed the access to more than 100,000 samples from over 20,000 patients treated at Policlinico Umberto I. It was possible to temporally investigate patient attendance patterns, average age distribution, seasonality of infections, and positivity rates of the analyzed viruses. RESULTS: Between 2019 and 2022 a significant reduction in organ transplants performed and in the positive molecular detection of EBV, JCV and BKV was observed. Additionally, there has been a noteworthy decrease in CMV reactivations, with a reduction of up to 50% starting in 2019. A remarkable reduction of 39% in the rate of CMV viral reactivation has been also achieved in SOT between 2016 and 2023. CONCLUSION: The years following 2019 were profoundly impacted by the COVID-19 pandemic era. This period resulted in a substantial reduction in healthcare services and hospital visits. Furthermore, the introduction of the drug Letermovir in Italy in 2019 demonstrated remarkable efficacy, evidenced by a reduction in CMV reactivations. Additionally, the adoption of a novel clinical approach centered on personalized therapy facilitated improved management of immunocompromised patients.

Establishment of COS-BK cells persistently infected with archetype BK polyomavirus.

BK polyomavirus (BKPyV) was the first human polyomavirus to be isolated from an immunosuppressed kidney transplant recipient in 1971. BKPyV reactivation causes BKPyV-associated nephropathy and hemorrhagic cystitis. However, the mechanisms underlying BKPyV replication remain unclear. In the present study, we performed the long-term cultivation of COS-7 cells transfected with archetype KOM-5 DNA, which were designated as COS-BK cells. BKPyV derived from COS-BK cells was characterized by analyzing the amount of the virus based on hemagglutination, viral replication, and the production of viral protein 1 (VP1). Immunostaining showed that VP1-positive cells accounted for a small percentage of COS-BK cells. The nucleotide sequences encompassing the origin of the DNA replication of BKPyV derived from COS-BK cells were generated from KOM-5 by the deletion of an 8-bp sequence, which did not involve T antigen binding sites. BKPyV replicated most efficiently in COS-BK cells in DMEM containing 2% fetal bovine serum. These results indicate that COS-BK cells are a suitable culture system for studying the persistent infection of archetype BKPyV.

The risk factors associated with post-transplantation BKPyV nephropathy and BKPyV DNAemia: a prospective study in kidney transplant recipients.

BACKGROUND: BK polyomavirus (BKPyV) infection after kidney transplantation can lead to serious complications such as BKPyV-associated nephropathy (BKPyVAN) and graft loss. The aim of this study was to investigate the incidence of BKPyVAN after implementing a BKPyV screening program, to map the distribution of BKPyV genotypes and subtypes in the Uppsala-Örebro region and to identify host and viral risk factors for clinically significant events. METHODS: This single-center prospective cohort study included kidney transplant patients aged ≥ 18 years at the Uppsala University Hospital in Sweden between 2016 and 2018. BKPyV DNA was analyzed in plasma and urine every 3 months until 18 months after transplantation. Also genotype and subtype were determined. A logistic regression model was used to analyze selected risk factors including recipient sex and age, AB0 incompatibility and rejection treatment prior to BKPyVAN or high-level BKPyV DNAemia. RESULTS: In total, 205 patients were included. Of these, 151 (73.7%) followed the screening protocol with 6 plasma samples, while184 (89.8%) were sampled at least 5 times. Ten (4.9%) patients developed biopsy confirmed BKPyVAN and 33 (16.1%) patients met criteria for high-level BKPyV DNAemia. Male sex (OR 2.85, p = 0.025) and age (OR 1.03 per year, p = 0.020) were identified as significant risk factors for developing BKPyVAN or high-level BKPyV DNAemia. BKPyVAN was associated with increased viral load at 3 months post transplantation (82,000 vs. < 400 copies/mL; p = 0.0029) and with transient, high-level DNAemia (n = 7 (27%); p < 0.0001). The most common genotypes were subtype Ib2 (n = 50 (65.8%)) and IVc2 (n = 20 (26.3%)). CONCLUSIONS: Male sex and increasing age are related to an increased risk of BKPyVAN or high-level BKPyV DNAemia. BKPyVAN is associated with transient, high-level DNAemia but no differences related to viral genotype were detected.

Detection of JC and BK polyomaviruses in patients with colorectal cancer (CRC) by PCR.

BACKGROUND: Overall, 20-30% of all cancers are estimated to be linked to infectious agents. Polyomaviruses are oncogenic cause in rodent models, readily transform their cells, and cause chromosomal instability in animal and human cells in-vitro. Some reports have indicated the presence of JCPyV and BKPyV in some human tumors. The JCPyV and BKPyV genome encodes some transforming proteins such as LT-Ag. Thus, these viruses could cause or promote some neoplasia, such as lymphomas, pancreatic, prostate, and colorectal cancers. Colorectal cancer (CRC) is the third most common cancer in the world. Risk factors for developing CRC are associated with personal features or habits, such as age, lifestyle, and gut microbiota. MATERIALS AND METHODS: In this study, we examined the prevalence of JCPyV and BKPyV in the 23 fecal samples of CRC patients and 24 healthy samples (control group). Virus DNA was extracted by a Favorgen DNA extraction kit. The large T antigen of JCPyV and VP1 of BKPyV were investigated by optimized multiplex PCR. RESULTS: One of the samples was positive for the JCPyV (4.3%), while in the samples of healthy individuals, the JCPyV was negative. Also, positive results for BKPyV PCR were obtained for five cases (21.7%) in the samples of the CRC group and one case (4.1%) in healthy individuals. CONCLUSION: The result showed no direct correlation between tumorigenesis and polyomavirus infections in CRC development. However, the exact role of BKPyV and JCPyV is still controversial and needs further study with larger sample size.

Shedding Light on Viral Shedding: Novel Insights into Nuclear Assembly, Cytoplasmic Transformation and Extracellular Vesicle Release of the BK Virus.

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.

Molecular Characterization of BK Polyomavirus Replication in Allogeneic Hematopoietic Cell Transplantation Patients.

BACKGROUND: High-level BK polyomavirus (BKPyV) replication in allogeneic hematopoietic cell transplantation (HCT) predicts failing immune control and BKPyV-associated hemorrhagic cystitis. METHODS: To identify molecular markers of BKPyV replication and disease, we scrutinized BKPyV DNA-loads in longitudinal urine and plasma pairs from 20 HCT patients using quantitative nucleic acid testing (QNAT), DNase-I treatment prior to QNAT, next-generation sequencing (NGS), and tested cell-mediated immunity. RESULTS: We found that larger QNAT amplicons led to under-quantification and false-negatives results (P < .001). DNase-I reduced urine and plasma BKPyV-loads by >90% (P < .001), indicating non-encapsidated BKPyV genomes. DNase-resistant urine BKPyV-loads remained infectious in cell culture. BKPyV genome fragmentation of ≤250 bp impaired NGS coverage of genetic variation using 1000-bp and 5000-bp amplicons. Conversely, 250-bp amplicons captured viral minority variants. We identified genotype-specific and genotype-independent changes in capsid Vp1 or T-antigen predicted to escape from antibody neutralization or cytotoxic CD8 T-cells, respectively. Genotype-specific changes in immunodominant 9mers were associated with reduced or absent CD8 T-cell responses. Thus, failure to control BKPyV replication in HCT Patients may involve insufficient genotype-specific cytotoxic CD8 T-cell responses, potentially predictable by low neutralizing antibodies as well as genotype-independent immune escape. CONCLUSIONS: Our results provide new insights for patient evaluation and for designing immune protection through neutralizing antibodies, adoptive T-cell therapy, or vaccines.

BK Polyomavirus Diversity After Hematopoietic Stem Cell Transplantation.

BK polyomavirus (BKPyV) infection is common after hematopoietic stem cell transplantation (HSCT) and is associated with the development of hemorrhagic cystitis (HC). The role that BKPyV plays in the pathogenesis of HC is not well characterized. We investigated the impact of BKPyV diversity on the development of HC using a previously established cohort of pediatric HSCT patients. There were 147 urine samples with quantifiable BKPyV at month 1 after HSCT; 137 (93.2%) were amplified using our in-house polymerase chain reaction approach and sent for next-generation sequencing. Subtype Ia was most frequent (61.3%), followed by subtype Ib1 (31.4%). The median viral load of subtype Ia samples was higher than for subtype Ib1 at month 1. Across the protein coding regions, APOBEC-induced mutations and signature patterns associated with HC were identified. This is the largest sequencing study of a single cohort of HSCT patients, providing a vast resource of sequence data for future analyses.

Dynamic viral integration patterns actively participate in the progression of BK polyomavirus-associated diseases after renal transplantation.

The classical lytic infection theory along with large T antigen-mediated oncogenesis cannot explain the BK polyomavirus (BKPyV)-associated tumor secondary to BKPyV-associated nephropathy (BKVAN), viremia/DNAemia, and viruria after renal transplantation. This study performed virome capture sequencing and pathological examination on regularly collected urine sediment and peripheral blood samples, and BKVAN and tumor biopsy tissues of 20 patients with BKPyV-associated diseases of different stages. In the early noncancerous stages, well-amplified integration sites were visualized by in situ polymerase chain reaction, simultaneously with BKPyV inclusion bodies and capsid protein expression. The integration intensity, the proportion of microhomology-mediated end-joining integration, and host PARP-1 and POLQ gene expression levels increased with disease progression. Furthermore, multiomics analysis was performed on BKPyV-associated urothelial carcinoma tissues, identifying tandem-like structures of BKPyV integration using long-read genome sequencing. The carcinogenicity of BKPyV integration was proven to disturb host gene expression and increase viral oncoprotein expression. Fallible DNA double-strand break repair pathways were significantly activated in the parenchyma of BKPyV-associated tumors. Olaparib showed an antitumor activity dose-response effect in the tumor organoids without BRCA1/2 genes mutation. In conclusion, the dynamic viral integration patterns actively participate in the progression of BKPyV-associated diseases and thus could be a potential target for disease monitoring and intervention.

BK Polyomavirus Requires the Mismatch Repair Pathway for DNA Damage Response Activation.

BK polyomavirus (PyV) infects the genitourinary tract of >90% of the adult population. Immunosuppression increases the risk of viral reactivation, making BKPyV a leading cause of graft failure in kidney transplant recipients. Polyomaviruses have a small double-stranded DNA (dsDNA) genome that requires host replication machinery to amplify the viral genome. Specifically, polyomaviruses promote S phase entry and delay S phase exit by activating the DNA damage response (DDR) pathway via an uncharacterized mechanism requiring viral replication. BKPyV infection elevates expression of MutSα, a mismatch repair (MMR) pathway protein complex that senses and repairs DNA mismatches and can activate the DDR. Thus, we investigated the role of the MMR pathway by silencing the MutSα component, Msh6, in BKPyV-infected primary cells. This resulted in severe DNA damage that correlated with weak DNA damage response activation and a failure to arrest the cell cycle to prevent mitotic entry during infection. Furthermore, silencing Msh6 expression resulted in significantly fewer infectious viral particles due to significantly lower levels of VP2, a minor capsid protein important for trafficking during subsequent infections. Since viral assembly occurs in the nucleus, our findings are consistent with a model in which entry into mitosis disrupts viral assembly due to nuclear envelope breakdown, which disperses VP2 throughout the cell, reducing its availability for encapsidation into viral particles. Thus, the MMR pathway may be required to activate the ATR (ATM-Rad3-related) pathway during infection to maintain a favorable environment for both viral replication and assembly. IMPORTANCE Since there are no therapeutics that target BKPyV reactivation in organ transplant patients, it is currently treated by decreasing immunosuppression to allow the natural immune system to fight the viral infection. Antivirals would significantly improve patient outcomes since reducing immunosuppression carries the risk of graft failure. PyVs activate the DDR, for which there are several promising inhibitors. However, a better understanding of how PyVs activate the DDR and what role the DDR plays during infection is needed. Here, we show that a component of the mismatch repair pathway is required for DDR activation during PyV infection. These findings show that the mismatch repair pathway is important for DDR activation during PyV infection and that inhibiting the DDR reduces viral titers by generating less infectious virions that lack the minor capsid protein VP2, which is important for viral trafficking.

Establishment and characterization of a novel reverse genetic system of BK polyomavirus.

BK polyomavirus (BKV) is a small non-enveloped DNA virus. BKV infection or reactivation may cause BKV-associated nephropathy and hemorrhagic cystitis in immunosuppressed transplant recipients. No effective antivirals or prevention strategies are available against BKV infections. The current BKV reverse system employs the transfection of purified full-length linear viral genomes released by enzyme digestion from BKV genomic plasmids. The method is laborious and often results in variable DNA yield and quality, which can affect the efficiency of transfection and subsequent formation of circular viral genomes in cells. In this study, we report the generation of circular viral genomes by Cre-mediated DNA recombination in cells directly transfected with BKV precursor genomic plasmids. The novel system supported efficient viral expression and replication, and produced a higher level of infectious virions compared with the transfection with linear BKV genomes. Furthermore, we successfully constructed recombinant BKV capable of reporter gene expression. In conclusion, the novel BKV reverse genetic system allows for simpler manipulation of BKV genome with better virus yield, providing a tool for the study of BKV life cycle and antiviral screening.

Detection of JCV or BKV viruria and viremia after kidney transplantation is not associated with unfavorable outcomes.

Studies analyzing the relationship between BK polyomavirus (BKV) or JC polyomavirus (JCV) infection and kidney transplant (KT) long term clinical outcomes are scarce. Therefore, we evaluated this relationship in a single-center retrospective cohort of 288 KT patients followed for 45.4(27.5; 62.5) months. Detection of BKV viremia in two consecutive analyses led to discontinuation of antimetabolite and initiation of mammalian target of rapamycin inhibitor. Outcome data included de novo BKV and/or JCV viremia and/or viruria after KT, death-censored graft survival and patient survival. BKV viruria and viremia were detected in 42.4% and 22.2% of KT recipients, respectively. BKV viremic patients had higher urinary BKV viral loads at the onset of viruria, when compared to nonviremic patients (7 log10 vs. 4.9 log10 cp/mL, p < 0.001). JCV viruria was identified in 38.5% of KT patients; the 5.9% of KT recipients who developed JCV viremia had higher JCV urinary viral loads at the onset of viruria, when compared to non-viremic patients (5.3 vs. 3.7 log10 cp/mL, p = 0.034). No differences were found in estimated glomerular filtration rate at the end of follow up, when comparing BKV or JCV viruric or viremic patients with nonviremic patients. No association was found between JCV or BKV viruria or viremia and death/graft failure. Therefore, higher BKV urinary viral loads at the onset could serve as an early maker of over immunosuppression. JCV and BKV replication was not associated with inferior clinical outcomes in KT patients with the above-mentioned immunosuppression strategy.

Histone deacetylase III interactions with BK polyomavirus large tumor antigen may affect protein stability.

BACKGROUND: Human polyomavirus BK (BKPyV) causes associated nephropathy and contributes to urinary tract cancer development in renal transplant recipients. Large tumor antigen (LT) is an early protein essential in the polyomavirus life cycle. Protein acetylation plays a critical role in regulating protein stability, so this study investigated the acetylation of the BKPyV LT protein. METHODS: The BKPyV LT nucleotide was synthesized, and the protein was expressed by transfection into permissive cells. The BKPyV LT protein was immunoprecipitated and subjected to LC-MS/MS analysis to determine the acetylation residues. The relative lysine was then mutated to arginine in the LT nucleotide and BKPyV genome to analyze the role of LT lysine acetylation in the BKPyV life cycle. RESULTS: BKPyV LT acetylation sites were identified at Lys3 and Lys230 by mass spectrometry. HDAC3 and HDAC8 and their deacetylation activity are required for BKPyV LT expression. In addition, mutations of Lys3 and Lys230 to arginine increased LT expression, and the interaction of HDAC3 and LT was confirmed by coimmunoprecipitation. CONCLUSIONS: HDAC3 is a newly identified protein that interacts with BKPyV LT, and LT acetylation plays a vital role in the BKPyV life cycle.

Infection by human polyomaviruses JCPyV and BKPyV in blood donors of Argentina.

BACKGROUND AND OBJECTIVES: A spectrum of blood-borne infectious agents may be transmitted through transfusion of blood components from asymptomatic donors. Despite the persistence of polyomaviruses in blood cells, no studies have been conducted in Argentina to assess the risk of transfusion infection. MATERIALS AND METHODS: We investigated BKPyV and JCPyV in 720 blood donors, using polymerase chain reaction (PCR) for a region of T antigen common to both viruses. Positive T-antigen samples were subjected to two additional PCR assays targeting the VP1 region. Viral genotypes were characterized by phylogenetic analysis. RESULTS: Polyomaviruses were detected in 1.25% (9/720) of the blood samples selected; JCPyV was identified in 0.97% (7/720) and BKPyV in 0.28% (2/720) of them. Phylogenetic analysis showed that the JCPyV sequences clustered with 2A genotype and Ia of BKPyV. CONCLUSION: This study describes for the first time the prevalence of polyomavirus DNA in blood donors of Córdoba, Argentina. The polyomavirus DNAemia in healthy populations suggests that those viruses are present in blood components eligible for transfusion. Therefore, the epidemiological surveillance of polyomavirus in blood banks might be incorporated into haemovigilance programmes, to determine the infectious risk and implement newer interventions to ensure the safety of blood supplies, if required.