Mapping of Human Polyomavirus in Renal Cell Carcinoma Tissues.

Worldwide, the incidence of renal cell carcinoma (RCC) is rising, accounting for approximately 2% of all cancer diagnoses and deaths. The etiology of RCC is still obscure. Here, we assessed the presence of HPyVs in paraffin-embedded tissue (FFPE) resected tissue from patients with RCC by using different molecular techniques. Fifty-five FFPE tissues from 11 RCC patients were included in this study. Consensus and HPyV-specific primers were used to screen for HPyVs. Both PCR approaches revealed that HPyV is frequently detected in the tissues of RCC kidney resections. A total of 78% (43/55) of the tissues tested were positive for at least one HPyV (i.e., MCPyV, HPyV6, HPyV7, BKPyV, JCPyV, or WUyV). Additionally, 25 tissues (45%) were positive for only one HPyV, 14 (25%) for two HPyVs, 3 (5%) for three HPyVs, and 1 one (1%) tissue specimen was positive for four HPyVs. Eleven (20%) RCC specimens were completely devoid of HPyV sequences. MCPyV was found in 24/55 RCC tissues, HPyV7 in 19, and HPyV6 in 8. The presence of MCPyV and HPyV6 was confirmed by specific FISH or RNA-ISH. In addition, we aimed to confirm HPyV gene expression by IHC. Our results strongly indicate that these HPyVs infect RCC and nontumor tissues, possibly indicating that kidney tissues serve as a reservoir for HPyV latency. Whether HPyVs possibly contribute to the etiopathogenesis of RCC remains to be elucidated.

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.

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.

BK polyomavirus infection: more than 50 years and still a threat to kidney transplant recipients.

BK polyomavirus (BKPyV) is a ubiquitous human polyomavirus and a major infection after kidney transplantation, primarily due to immunosuppression. BKPyV reactivation can manifest as viruria in 30%-40%, viremia in 10%-20%, and BK polyomavirus-associated nephropathy (BKPyVAN) in 1%-10% of recipients. BKPyVAN is an important cause of kidney graft failure. Although the first case of BKPyV was identified in 1971, progress in its management has been limited. Specifically, there is no safe and effective antiviral agent or vaccine to treat or prevent the infection. Even in the current era, the mainstay approach to BKPyV is a reduction in immunosuppression, which is also limited by safety (risk of de novo donor specific antibody and rejection) and efficacy (graft failure). However, recently BKPyV has been getting more attention in the field, and some new treatment strategies including the utilization of viral-specific T-cell therapy are emerging. Given all these challenges, the primary focus of this article is complications associated with BKPyV, as well as strategies to mitigate negative outcomes.

Belatacept-based immunosuppression does not confer increased risk of BK polyomavirus-DNAemia relative to tacrolimus-based immunosuppression.

BACKGROUND: The effect of belatacept on BK polyomavirus (BKPyV) control remains largely unknown. METHODS: This is a propensity matched retrospective cohort study in adult kidney transplant recipients (KTR) transplanted between 2016-2020 who received a belatacept- versus tacrolimus-based immunosuppression regimen. A continuous time multi-state Markov model was used to evaluate BKPyV replication dynamics (BKPyV-dyn). Three BKPyV-dyn states were defined: BKPyV-dyn1 (viral load <3 log10), BKPyV-dyn2 (viral load ≥ 3 log10 and ≤4 log10), and BKPyV-dyn3 (viral load >4 log10). RESULTS: Two hundred eighty KTR on belatacept- and 280 KTR on tacrolimus-based regimens were compared. The probability of transitioning between BKPyV-dyn states and time spent in each state in both groups was comparable. Total duration in BKPyV-dyn-1 was 632.1 days (95% CI 612.1, 648.5) for belatacept versus 615.2 days (95% CI 592.5, 635.8) for tacrolimus, BKPyV-dyn-2 was 49.2 days (95% CI 41.3, 58.4) for belatacept versus 55.6 days (95% CI 46.5, 66.8) for tacrolimus, and BKPyV-dyn-3 was 48.7 days (95% CI 37.1, 363.1) for belatacept versus 59.2 days (95% CI 45.8, 73.5) for tacrolimus. BKPyV associated nephropathy (PyVAN) occurred in 3.9% in belatacept- and 3.9% tacrolimus-treated KRT (P > .9). CONCLUSIONS: Compared with tacrolimus-based immunosuppression, belatacept based immunosuppression was not associated with increased risk of BKPyV-DNAemia or nephropathy.

Comparative performance evaluation of random access and real-time PCR techniques in the diagnosis of BK virus infections in transplant patients.

PURPOSE: The study aims to compare random-access NeuMoDx values with artus qPCR values to validate the accuracy of NeuMoDx as an alternative to qPCR and provide an equation to convert copies/ml to IU/ml measurements. METHODS: A total of 95 samples, including 61 transplant patient samples (n = 23 urine, n = 38 plasma) as the study group, 28 BKPyV-free samples as the control group, and six quality control samples, were included. One-Way ANOVA, Pearson correlation, Bland-Altman, Passing-Bablok, Deming regression analyses were used for statistical evaluation. RESULTS: Of 95 samples, 46 (48 %) were positive with NeuMoDx, while 40 (42 %) were positive with artus qPCR. Both techniques were statistically similar (p > 0.05). Deming correlation analysis (r = 0.9590), Passing Bablok and Bland Altman analyses demonstrated a strong correlation between NeuMoDx and artus values. The equation that provides the conversion between NeuMoDx and artus qPCR values was NeuMoDx= (1.12965 x artus qPCR) - 0.55016. BKPyV infections remain a concern for transplant patients globally, and effective new diagnostic methods are required. CONCLUSIONS: Consistency between the results of NeuMoDx and qPCR confirms that NeuMoDx may be a valuable alternative for detecting BKPyV to prevent viral propagation. Our findings may allow converting copy/ml results to IU/ml for diagnosing and monitoring BKPyV infections in transplant patients.

The Conserved YPX3L Motif in the BK Polyomavirus VP1 Protein Is Important for Viral Particle Assembly but Not for Its Secretion into Extracellular Vesicles.

The BK polyomavirus (BKPyV) is a small DNA non-enveloped virus whose infection is asymptomatic in most of the world's adult population. However, in cases of immunosuppression, the reactivation of the virus can cause various complications, and in particular, nephropathies in kidney transplant recipients or hemorrhagic cystitis in bone marrow transplant recipients. Recently, it was demonstrated that BKPyV virions can use extracellular vesicles to collectively traffic in and out of cells, thus exiting producing cells without cell lysis and entering target cells by diversified entry routes. By a comparison to other naked viruses, we investigated the possibility that BKPyV virions recruit the Endosomal-Sorting Complexes Required for Transport (ESCRT) machinery through late domains in order to hijack extracellular vesicles. We identified a single potential late domain in the BKPyV structural proteins, a YPX3L motif in the VP1 protein, and used pseudovirions to study the effect of point mutations found in a BKPyV clinical isolate or known to ablate the interaction of such a domain with the ESCRT machinery. Our results suggest that this domain is not involved in BKPyV association with extracellular vesicles but is crucial for capsomere interaction and thus viral particle assembly.

Nonclinical and clinical characterization of MAU868, a novel human-derived monoclonal neutralizing antibody targeting BK polyomavirus VP1.

Reactivation of BK polyomavirus (BKPyV) can cause significant kidney and bladder disease in immunocompromised patients. There are currently no effective, BKPyV-specific therapies. MAU868 is a novel, human immunoglobulin (Ig) G1 monoclonal antibody that binds the major capsid protein, VP1, of BKPyV with picomolar affinity, neutralizes infection by the 4 major BKPyV genotypes (EC50 ranging from 0.009-0.093 µg/mL; EC90 ranging from 0.102-4.160 µg/mL), and has comparable activity against variants with highly prevalent VP1 polymorphisms. No resistance-associated variants were identified in long-term selection studies, indicating a high in vitro barrier-to-resistance. The high-resolution crystal structure of MAU868 in complex with VP1 pentamer identified 3 key contact residues in VP1 (Y169, R170, and K172). A first-in-human study was conducted to assess the safety, tolerability, and pharmacokinetics of MAU868 following intravenous and subcutaneous administration to healthy adults in a randomized, placebo-controlled, double-blinded, single ascending dose design. MAU868 was safe and well-tolerated. All adverse events were grade 1 and resolved. The pharmacokinetics of MAU868 was typical of a human IgG, with dose-proportional systemic exposure and an elimination half-life ranging between 23 and 30 days. These results demonstrate the potential of MAU868 as a first-in-class therapeutic agent for the treatment or prevention of BKPyV disease.

BK Polyomavirus in Pediatric Renal Transplantation-What We Know and What We Do Not.

BK polyomavirus (BKPyV) is still a real threat in the management of kidney transplantation. Immunosuppressive treatment disrupts the equilibrium between virus replication and immune response, and uncontrolled BKPyV replication leads to nephropathy (BKPyV nephropathy). The first evidence of BKPyV reactivation in transplant recipients is the detection of viral shedding in urine, which appears in 20% to 60% of patients, followed by BKPyV viremia in 10-20% of kidney transplant recipients. BKPyV nephropathy eventually occurs in 1-10% of this population, mainly within the first 2 years post-transplantation, causing graft loss in about half of those patients. Few data exist regarding the pediatric population and we focus on them. In this paper, we review the existing diagnostic methods and summarize the evidence on the role of BKPyV humoral and cellular immunity in modulating the clinical course of BKPyV infection and as potential predictors of the outcome. We look at the known risk factors for BKPyV nephropathy in the immunosuppressed patient. Finally, we propose a sensible clinical attitude in order to screen and manage BKPyV infection in kidney transplant children.

Epidemiology and Dynamics of BK Polyomavirus Replication after Kidney Transplantation.

BACKGROUND/OBJECTIVES: In the absence of an effective antiviral treatment for BK polyomavirus (BKPyV), a better understanding of the epidemiology and time course of BKPyV replication after kidney transplantation is needed to limit the virus's impact on the graft outcome. METHODS: In a 7-year study, we screened more than 430 kidney transplant recipients and analyzed the time course and virological characteristics of BKPyV replication. RESULTS: Urinary viral replication was observed in 116 (27%) of the 430 patients, and 90 of the 116 (78%) had viral DNAemia. Thirty-eight patients (8.8%) were presumed to have nephropathy (DNAemia > 4 log10 copies/mL). Of the patients with BKPyV replication, 48%, 60%, 71%, and 80% were first found to be positive one, two, three, and four months post-transplantation. The initial viral load in the urine was below 7 log10 copies/mL in 100% of the patients with viral replication first detected before the first month, and this proportion was 57% when viral replication was first detected after the first month. When the BKPyV replication was first detected in a urine sample at month 3 or later, 81.5% of patients had concomitant BKPyV DNAemia. The predominant viral subtype was Ib2 (60%), and there was no apparent relationship between the subtype and the time course of BKPyV replication. CONCLUSIONS: Urinary BKPyV replication occurs early after renal transplantation and in most patients will increase to a level requiring therapeutic intervention. Close monitoring for BKPyV in the early post-transplantation period would enable the pre-emptive adjustment of the immunosuppression regimen.

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.

Longitudinal study of human polyomaviruses viruria in kidney transplant recipients.

INTRODUCTION: Immunosuppression after kidney transplantation (KTx) exposes recipients to Human Polyomaviruses (HPyVs) infections, whose natural history is still misunderstood. METHODS: Allograft biopsies, and urine from 58 donor-recipient pairs were collected before KTx (T0) and 1 (T1), 15 (T2), 30 (T3), 60 (T4), 90 (T5), 180 (T6), 270 (T7), 360 (T8), and 540 (T9) days after transplant. Specimens were tested for JC (JCPyV) and BK (BKPyV), by quantitative Real-Time PCR. The course of post-KTx HPyVs viruria, and the association between JCPyV viruria in recipients and donors, were evaluated. RESULTS: HPyVs were detected in 3/58 (5.2%) allograft biopsies. HPyVs viruria was present in 29/58 (50%) donors and 41/58 (70.7%) recipients. JCPyV DNA was detected in 26/58 (44.8%) donors and 25/58 recipients (43.1%), 19 of whom received kidney from JCPyV positive donor, whereas BKPyV genome was detected in 3 (5.2%) donors and 22 (37.9%) recipients. The median time of JCPyV, and BKPyV first episode of replication was 1, and 171 days post KTx, respectively. At T0, JCPyV viruria of donors was associated with increased risk of JCPyV replication post-KTx; recipients with JCPyV positive donors showed lower risk of BKPyV replication post-KTx. CONCLUSIONS: The results suggested that JCPyV may be transmitted by allograft, and that its replication post KTx might prevent BKPyV reactivation. Future investigation regarding correlation between chronic exposure to immunosuppressive agents and HPyVs urinary replication are warranted.

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.

The relation of NCCR variations and host transcription factors gene regulation in BK polyomavirus infected kidney transplant patients.

BACKGROUND: BK polyomavirus (BKPyV) infection in immunocompromised patients can led to polyomavirus-associated nephropathy (BKPyVAN) especially after kidney transplantation. The polyomavirus genome contains enhancer elements that are important transcription activators. In this study, the association between viral and host gene expression and NCCR variations was evaluated in kidney transplant recipients (KTRs) with BKPyV active, and BKPyV in-active infection. METHODS AND RESULTS: Blood samples were collected from selected KTRs who divided to patients with active and in-active BKPyV infection. Transcriptional control region (TCR) anatomy was compared to the genomic sequence of archetype BKPyV strain WW using nested PCR method and sequencing. The expression level of some transcription factor genes was evaluated using in-house Real-time PCR (SYBR Green) technique. Most changes were observed after TCR anatomy detection in the Q and P blocks. The expression level of VP1 and LT-Ag viral genes were significantly higher in patients with active infection compared with non-infected ones. Transcription factor genes SP1, NF1, SMAD, NFκB, P53, PEA3, ETS1, AP2, NFAT and AP1 were significantly higher in BKPyV active group in comparison in-active and control groups. The analyses revealed that viral load level and mutations frequency has significant correlation. CONCLUSIONS: Based on the results, increasing of NCCR variations were associated with higher viral load of BKPyV especially in Q block. Host transcriptional factors and viral genes all had higher express level in active BKPyV patients versus no in-active ones. Detection of the relation between NCCR variation and BKPyV severity in KTRs need to be confirmed in further complicated studies.

Detection of BK and JC polyomaviruses in sewage water of the urban areas of Lahore, Pakistan.

The surveillance of sewage water has become an extremely essential tool to trace the circulation of viruses in a population and to predict the outbreak of viral diseases. Sewage monitoring is important for those viruses which cause subclinical infections since it is difficult to determine their prevalence. Polyomaviruses are ubiquitously present, circular double-stranded DNA viruses that can infect humans as well. Among all human polyomaviruses, BK polyomavirus and JC polyomavirus associated with the development of aggressive diseases in immunocompromised individuals, are highly prevalent. This study aimed to investigate the presence and the quantitative prevalence of these two disease-associated human polyomaviruses in sewage water collected from six drains of Lahore, Pakistan. The viruses present in the environmental samples were concentrated by PEG method before isolating viral nucleic acids. Conventional PCR amplifications were performed for molecular detection of BK polyomavirus and JC polyomavirus targeting their large tumor antigen genetic region. The presence of BK polyomavirus and JC polyomavirus was confirmed in the DNA extracted from concentrated sewage samples of each drain by performing both qualitative and quantitative PCR. Our data shows that the viral load ranged from 1278 to 178368 copies per µg of environmental DNA for BK polyomavirus and 5173 to 79129 copies per µg of environmental DNA for JC polyomavirus. In conclusion, here we report first time the detection of BK polyomavirus and JC polyomavirus in sewage water collected from six main drains in urban areas of Lahore, Pakistan showing the high prevalence of these viruses in the Pakistani population. This assay could be used as a proxy to determine the prevalence of these viruses in the Pakistani population.

Single-Cell, High-Content Microscopy Analysis of BK Polyomavirus Infection.

By adulthood, the majority of the population is persistently infected with BK polyomavirus (BKPyV). Only a subset of the population, generally transplant recipients on immunosuppressive drugs, will experience disease from BKPyV, but those who do have few treatment options and, frequently, poor outcomes, because to date there are no effective antivirals to treat or approved vaccines to prevent BKPyV. Most studies of BKPyV have been performed on bulk populations of cells, and the dynamics of infection at single-cell resolution have not been explored. As a result, much of our knowledge is based upon the assumption that all cells within a greater population are behaving the same way with respect to infection. The present study examines BKPyV infection on a single-cell level using high-content microscopy to measure and analyze the viral protein large T antigen (TAg), promyelocytic leukemia protein (PML), DNA, and nuclear morphological features. We observed significant heterogeneity among infected cells, within and across time points. We found that the levels of TAg within individual cells did not necessarily increase with time and that cells with the same TAg levels varied in other ways. Overall, high-content, single-cell microscopy is a novel approach to studying BKPyV that enables experimental insight into the heterogenous nature of the infection. IMPORTANCE BK polyomavirus (BKPyV) is a human pathogen that infects nearly everyone by adulthood and persists throughout a person's life. Only people with significant immune suppression develop disease from the virus, however. Until recently the only practical means of studying many viral infections was to infect a group of cells in the laboratory and measure the outcomes in that group. However, interpreting these bulk population experiments requires the assumption that infection influences all cells within a group similarly. This assumption has not held for multiple viruses tested so far. Our study establishes a novel single-cell microscopy assay for BKPyV infection. Using this assay, we discovered differences among individual infected cells that have not been apparent in bulk population studies. The knowledge gained in this study and the potential for future use demonstrate the power of this assay as a tool for understanding the biology of BKPyV.

Incidence, Risk Factors, and Outcomes of Kidney Transplant Recipients With BK Polyomavirus-Associated Nephropathy.

Introduction: BK polyomavirus-associated nephropathy (BKPyVAN) is associated with graft dysfunction and loss; however, knowledge of immunosuppression reduction strategies and long-term graft, and patient outcomes across the disease spectrum is lacking. Methods: This cohort study included 14,697 kidney transplant recipients in Australia and New Zealand (2005-2019), followed for 91,306 person years. Results: BKPyVAN occurred in 460 recipients (3%) at a median posttransplant time of 4.8 months (interquartile range, 3.1-10.8). Graft loss (35% vs. 21%, P < 0.001), rejection (42% vs. 25%, P < 0.001), and death (18% vs. 13%, P = 0.002) were more common in the BKPyVAN group. The most frequent changes in immunosuppression after BKPyVAN were reduction (≤50%) in tacrolimus (172, 51%) and mycophenolate doses (134, 40%), followed by the conversion of mycophenolate to leflunomide (62, 19%) and tacrolimus to ciclosporin (20, 6%). Factors associated with the development of BKPyVAN included (adjusted hazard ratio [HR]; 95% confidence interval) male sex (1.66; 1.34-2.05), recipient age (≥70 vs. <20 [2.46; 1.30-4.65]), recipient blood group (A vs. B [2.00; 1.19-3.34]), donor age (≥70 vs. <20 [2.99; 1.71-5.22]), earlier era (1.74; 1.35-2.25), donor/recipient ethnic mismatch (1.52; 1.23-1.87), tacrolimus use (1.46; 1.11-1.91), and transplantation at a lower-volume transplant center (1.61; 1.24-2.09). The development of BKPyVAN was associated with an increased risk of all-cause (1.75; 1.46-2.09) and death-censored graft loss (2.49; 1.99-3.11), but not mortality (1.15; 0.91-1.45). Conclusions: BKPyVAN is associated with an increased risk of all-cause and death-censored graft loss, but not death. Interventional trials are urgently needed to evaluate the efficacy of immunosuppression reduction and novel strategies to minimize the adverse outcomes associated with BKPyVAN.

The role of the DE and EF loop of BKPyV VP1 in the serological cross-reactivity between subtypes.

BK virus (BKPyV) is a causative agent of BKPyV-associated nephropathy and graft rejections in kidney transplant patients. It establishes persistent infection in the kidneys, which can lead to reactivation in an immunosuppressed state or transmission to kidney recipients. Complications in the case of donor-derived infections can be caused by differences between the four known BKPyV subtypes, as prior infection with one subtype does not guarantee protection against de novo infection with other subtypes. The recipient and donor pretransplant serotyping is not routinely performed since simple ELISA tests employing antigens derived from the major viral capsid protein 1 (VP1) are hindered by the high cross-reactivity of anti-VP1 antibodies against all subtypes. Identifying subtype-specific epitopes in VP1 could lead to the design of specific antigens and the improvement of serodiagnostics for kidney transplantation. We aimed to study the surface residues responsible for the interactions with the subtype-specific antibodies by focusing on the DE and EF loops of VP1, which have only a small number of distinct amino acid differences between the most common subtypes, BKPyV-I and BKPyV-IV. We designed two mutant virus-like particles (VLPs): we introduced BKPyV-I characteristic amino acid residues (either H139N in the DE loop or D175E and I178V changes in the EF loop) into the base sequence of a BKPyV-IV VP1. This way, we created BKPyV-IV mutant VLPs with the sequence of either the BKPyV-I DE loop or the BKPyV-I EF loop. These mutants were then used as competing antigens in an antigen competition assay with a panel of patient sera, and changes in antibody reactivity were assessed by ELISA. We found that the changes introduced into the BKPyV-IV VP1 EF loop restrict antibody recognition in most samples and that converting the BKPyV-IV DE loop into its BKPyV-I equivalent attracts anti-VP1 BKPyV-I antibodies. Although our results did not lead to the discovery of a subtype-specific epitope on the VP1, they suggested that the arrangement of the EF loop in VP1 might dictate the mode of interaction between virus and anti-VP1 antibodies in general and that the interactions between the antibodies and the viral capsid might be very complex. Consequently, an antigen competition assay as an assay to distinguish between BKPyV serotypes might prove difficult to interpret.

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.