Human polyoma virus BK DNA detection by nested PCR in renal transplant recipients

Several studies report a correlation between the human polyomavirus BK (BKV) and interstitial nephritis in renal transplant recipients in whom immunosuppressive treatment is thought to allow or induce reactivation of the virus. Furthermore, it is described that nephropathy may result from the use of newly introduced immunosuppressive drugs. In the present study, we evaluated the presence of BKV DNA by nested-PCR (n-PCR) in urine and serum samples from 35 renal transplant patients related to the immunosuppressive regimens and renal function.     

Prospective study of urine cytology screening for BK polyoma virus replication in renal transplant recipients

Objective:  BK virus (BKV) may be associated with interstitial nephritis in renal transplant recipients and this can lead to irreversible chronic allograft dysfunction. Early diagnosis of BKV nephropathy determines its progress because no specific antiviral therapy exists. Urine cytology, detection of viral DNA in urine or blood and renal biopsy are the main diagnostic tools. The purpose of this study was to evaluate the use of urine cytology for diagnosis of BKV replication in renal graft recipients.
Patients and methods:  We studied 32 de novo renal transplant recipients prospectively with sequential urine samples for a period of 1 year. Thin-Prep methodology was used to prepare the slides. Cytology results were correlated with polymerase chain reaction (PCR) in urine and blood.
Results:  Decoy cells indicative of BKV infection were detected in 14 (7.3%) of the 190 urine samples derived from 11 recipients. In three cases with positive decoy cells, BK viraemia and viruria were simultaneously identified. In a further three cases, BKV active replication was confirmed in urine by both cytology and PCR.
Conclusions:  Urine cytology is an easy and rapid method of detecting decoy cells in cases where renal biopsy is not possible. However, the low incidence of detection of decoy cells in the present study, together with poor correlation with PCR results, questions its sensitivity and specificity in diagnosing BKV reactivation.     

BK virus DNA detection by real-time polymerase chain reaction in clinical specimens

The BK polyomavirus (BKV) is widespread in the general population. In transplant recipients, the patients' weakened immune response may encourage reactivation of latent infection, leading to BKV-related diseases. Rapid and quantitative detection might help to delineate viral reactivation patterns and could thus play an important role in their clinical management. In our study we developed an "in-house" quantitative real-time PCR to detect BKV DNA. The effectiveness of this assay was evaluated by a retrospective analysis of 118 plasma specimens from 22 bone marrow transplant (BMT) recipients and 107 samples from immunocompetent subjects. Eight (36.3%) of the 22 bone marrow transplant recipients tested positive for BKV. The viral load varied from specimen to specimen (10 to 10(5) copies/ml). BKV related disease like hemorrhagic cystitis (HC) was diagnosed in three patients. Specimens from the control group all tested negative. Our results showed the high sensitivity of the real-time PCR, allowing accurate and reproducible measuring of the viral load in order to identify patients at risk for BKV-related diseases. With due caution in interpreting threshold values, the real-time PCR could provide a rapid, sensitive and specific tool for detecting BKV and distinguishing latent and active infection.     

An N-linked glycoprotein with alpha(2,3)-linked sialic acid is a receptor for BK virus

BK virus (BKV) is a common human polyomavirus infecting >80% of the population worldwide. Infection with BKV is asymptomatic, but reactivation in renal transplant recipients can lead to polyomavirus-associated nephropathy. In this report, we show that enzymatic removal of alpha(2,3)-linked sialic acid from cells inhibited BKV infection. Reconstitution of asialo cells with alpha(2,3)-specific sialyltransferase restored susceptibility to infection. Inhibition of N-linked glycosylation with tunicamycin reduced infection, but inhibition of O-linked glycosylation did not. An O-linked-specific alpha(2,3)-sialyltransferase was unable to restore infection in asialo cells. Taken together, these data indicate that an N-linked glycoprotein containing alpha(2,3)-linked sialic acid is a critical component of the cellular receptor for BKV.     

Infection of vero cells by BK virus is dependent on caveolae

Polyomavirus-associated nephropathy occurs in approximately 5% of renal transplant recipients and results in loss of graft function in 50 to 70% of these patients. The disease is caused by reactivation of the common human polyomavirus BK (BKV) in the transplanted kidney. The early events in productive BKV infection are unknown. In this report, we focus on elucidating the mechanisms of BKV internalization in its target cell. Our data reveal that BKV entry into permissive Vero cells is slow, is independent of clathrin-coated-pit assembly, is dependent on an intact caveolin-1 scaffolding domain, is sensitive to tyrosine kinase inhibition, and requires cholesterol. BKV colocalizes with the caveola-mediated endocytic marker cholera toxin subunit B but not with the clathrin-dependent endocytic marker transferrin. In addition, BKV infectious entry is sensitive to elevation in intracellular pH. These findings indicate that BKV entry into Vero cells occurs by caveola-mediated endocytosis involving a pH-dependent step.     

Detection of BK virus in urine from renal transplant subjects by mass spectrometry

Background

The diagnosis and management of BK virus (BKV) reactivation following renal transplantation continues to be a significant clinical problem. Following reactivation of latent virus, impaired cellular immunity enables sustained viral replication to occur in urothelial cells, which potentially leads to the development of BKV-associated nephropathy (BKVAN). Current guidelines recommend regular surveillance for BKV reactivation through the detection of infected urothelial cells in urine (decoy cells) or viral nucleic acid in urine or blood. However, these methods have variable sensitivity and cannot routinely distinguish between different viral subtypes. We therefore asked whether mass spectrometry might be able to overcome these limitations and provide an additional non-invasive technique for the surveillance of BKV and identification of recipients at increased risk of BKVAN.

Results

Here we describe a mass spectrometry (MS)-based method for the detection of BKV derived proteins directly isolated from clinical urine samples. Peptides detected by MS derived from Viral Protein 1 (VP1) allowed differentiation between subtypes I and IV. Using this approach, we observed an association between higher decoy cell numbers and the presence of the VP1 subtype Ib-2 in urine samples derived from a cohort of 20 renal transplant recipients, consistent with the hypothesis that certain viral subtypes may be associated with more severe BKVAN.

Conclusions

This is the first study to identify BK virus proteins in clinical samples by MS and that this approach makes it possible to distinguish between different viral subtypes. Further studies are required to establish whether this information could lead to stratification of patients at risk of BKVAN, facilitate distinction between BKVAN and acute rejection (AR), and ultimately improve patient treatment and outcomes.     

BK virus-associated infection in cerebrospinal fluid of neurological patients and mutation analysis of the complete VP1 gene in different patient groups

While BK virus (BKV) is frequently associated with pathological conditions in bone marrow and renal transplant recipients, BKV infection in neurological individuals has been rarely reported. As a result of a BKV, JCV, and SV40 large T antigen-specific multiplex PCR on 2,062 cerebrospinal fluid (CSF) samples from neurological patients suspicious of JCV infection, we identified 20 subjects with at least 1 CSF specimen positive for BKV large T antigen DNA. Because VP1 protein has been suggested to influence the biological/pathological properties of BKV, we tried to sequence the entire VP1 gene in the BKV-positive neurological patients and succeeded in 14 of the 20 neurological patients. To compare the VP1 sequence of the BKV neurological strains with that of non-neurotropic strains in other clinical situations, full-length VP1 DNA was sequenced in 15 renal and 6 bone marrow transplant recipients positive to BKV-viremia, and in 8 pregnant women as non-pathological controls. An increased (respectively, decreased) tendency for mutations in the BC loop (respectively, EF loop) was observed, and no mutations were detected in the CD, GH, and HI loops. Subtype I was predominant (93%) and compared to archetypal BKV (WW), amino acid substitutions were detected in 4/14 neurological patients, 10/15 renal transplant recipients, 3/6 bone marrow transplant patients, and in all the pregnant women. Each patient group had distinctive VP1 mutations, but these unique substitutions were not present in all patients of this group. However, molecular modeling simulations of the VP1 mutants predicted changes in protein surface properties which might affect the VP1-receptor interaction.     

The human polyomavirus BK: Potential role in cancer

In human cancer, a role has been suggested for the human polyomavirus BK, primarily associated with tubulointerstitial nephritis and ureteric stenosis in renal transplant recipients, and with hemorrhagic cystitis in bone marrow transplant (BMT) recipients. After the initial infection, primarily unapparent and without clinical signs, the virus disseminates and establishes a persistent infection in the urinary tract and lymphocytes. There is correlative evidence regarding potential role of polyomavirus BK in cancer. In fact, the BK virus (BKV) DNA (complete genome and/or subgenomic fragments containing the early region) is able to transform embryonic fibroblasts and cells cultured from kidney and brain of hamster, mouse, rat, rabbit, and monkey. Nevertheless, transformation of human cells by BKV is inefficient and often abortive. Evidence supporting a possible role for BKV in human cancer has accumulated slowly in recent years, after the advent of polymerase chain reaction (PCR). BKV is known to commonly establish persistent infections in people and to be excreted in the urine by individuals who are asymptomatic, complicating the evaluation of its potential role in development of human cancer. Therefore, there is no certain proof that human polyomavirus BK directly causes the cancer in humans or acts as a cofactor in the pathogenesis of some types of human cancer.     

BK antibody and virus-specific IgM responses in renal transplant recipients,patients with malignant disease,and healthy people.

Haemagglutination-inhibition (HAI) antibodies to BK virus, including BK-virus-specific IgM, were determined before and after renal transplantation in 20 patients, in 57 patients with malignant disease, and in 66 healthy controls, Before transplantation 11 of the renal transplant recipients were seronegative, but eight later serocconverted, two before and six after transplantation. Twenty of the patients with malignant disease and 22 controls were also seronegative. The geometric mean titre of BK HAI antibodies was significantly higher among transplanted patients (1/180) than among controls (1/90). BK-virus-specific IgM antibody was detected in seven renal transplant recipients, six patients with malignant disease, and 13 healthy controls. In transplant recipients BK-virus-specific IgM antibody usually persisted throughout the duration of the study, and studies on controls from whom second serum samples were available suggested that they too had persistent BK-virus-specific IgM responses. The geometric mean titre of BK-virus-specific IgM HAI antibody was significantly greater in post-transplantation sera (1/223) than in control sera (1/28). The specificity of the detection of BK-virus-specific IgM HAI antibody was confirmed by direct visualisation of antibody by immune electron microscopy. The persistence of BK-virus-specific IgM suggested that BK virus continued to provide an antigenic stimulus. Nevertheless, there was no obvious association between the serological findings and any clinical features, and prospective studies will be needed to elucidate any such association.     

Real-Time RT-PCR Assay for the Quantitation of Polyomavirus BK VP1 mRNA Levels in Urine

In renal transplant recipients, polyomavirus BK can reactivate resulting in graft nephropathy. Screening for BK virus replication may allow for earlier interventions with reduced allograft loss. The measurement of urinary cell BKV VP1 mRNA for identify viral replication levels at risk of developing nephropathy has been proposed. In this article, the development, optimization, and standardization of a Taqman real-time RT-PCR assay for the quantitation of BKV VP1 mRNA levels in urine is described. Subsequently, the method has been validated on urine specimens obtained from renal transplant recipients. The use of VP1 mRNA measurement as a marker for viral replication and a tool for noninvasive diagnosis of nephropathy should be regarded with great caution, given the potentially limited positive predictive value and the drawbacks associated with the complexity of the real-time RT-PCR assay requiring an expert well trained operator and the relatively poor cost-efficiency ratio.     

BK Polyomavirus Genotypes Represent Distinct Serotypes with Distinct Entry Tropism

BK polyomavirus (BKV) causes significant urinary tract pathogenesis in immunosuppressed individuals, including kidney and bone marrow transplant recipients. It is currently unclear whether BKV-neutralizing antibodies can moderate or prevent BKV disease. We developed reporter pseudoviruses based on seven divergent BKV isolates and performed neutralization assays on sera from healthy human subjects. The results demonstrate that BKV genotypes I, II, III, and IV are fully distinct serotypes. While nearly all healthy subjects had BKV genotype I-neutralizing antibodies, a majority of subjects did not detectably neutralize genotype III or IV. Surprisingly, BKV subgenotypes Ib1 and Ib2 can behave as fully distinct serotypes. This difference is governed by as few as two residues adjacent to the cellular glycan receptor-binding site on the virion surface. Serological analysis of mice given virus-like particle (VLP)-based BKV vaccines confirmed these findings. Mice administered a multivalent VLP vaccine showed high-titer serum antibody responses that potently cross-neutralized all tested BKV genotypes. Interestingly, each of the neutralization serotypes bound a distinct spectrum of cell surface receptors, suggesting a possible connection between escape from recognition by neutralizing antibodies and cellular attachment mechanisms. The finding implies that different BKV genotypes have different cellular tropisms and pathogenic potentials in vivo. Individuals who are infected with one BKV serotype may remain humorally vulnerable to other BKV serotypes after implementation of T cell immunosuppression. Thus, prevaccinating organ transplant recipients with a multivalent BKV VLP vaccine might reduce the risk of developing posttransplant BKV disease.     

BKV-DNA and JCV-DNA Co-quantification assay to evaluate viral load in urine and serum

Infections from human polyomaviruses BK and JC (BKV and JCV) occur independently, but concomitant infections and the simultaneous persistence of both viruses have been observed in renal transplant recipients. Several studies have disclosed a correlation between BKV and interstitial nephritis in renal transplant recipients, and an association between JCV and some cases of nephropathy has recently been hypothesized. This article describes the development of a semiquantitative-nested polymerase chain reaction (PCR) assay to simultaneously detect BKV and JCV viral load in urine and serum. The first-round amplification step uses primers that amplify a 385-bp DNA fragment from the “large T antigen” region of both viruses. Samples testing positive in the first step are then run in the second step. In the second-round amplification, different inner primers are used to separately quantify BKV-DNA and/or JCV-DNA. The assay offers several advantages including: (1) rapid submission of clinical samples to screening; (2) verification of the absence of Taq polymerase inhibitors with the use of an internal control; (3) a sensitivity threshold of 10 copies/reaction; and (4) assay running is less labor intensive, cheap, and easy to perform. The assay may be easily used to monitor viral loads versus baseline levels in urine and serum samples from renal transplant recipients to detect those at risk of BKV- or JCV-related nephropathy, and to monitor their response to immunosuppression reduction therapy if it occurs. This paper is dedicated to the memory of Prof. Giorgio Cavallo.     

Interplay of cellular and humoral immune responses against BK virus in kidney transplant recipients with polyomavirus nephropathy

Reactivation of the polyomavirus BK (BKV) causes polyomavirus nephropathy (PVN) in kidney transplant (KTx) recipients and may lead to loss of the renal allograft. We have identified two HLA-A*0201-restricted nine-amino-acid cytotoxic T lymphocyte (CTL) epitopes of the BKV major capsid protein VP1, VP1(p44), and VP1(p108). Using tetramer staining assays, we showed that these epitopes were recognized by CTLs in 8 of 10 (VP1(p44)) and 5 of 10 (VP1(p108)) HLA-A*0201+ healthy individuals, while both epitopes elicited a CTL response in 10 of 10 KTx recipients with biopsy-proven PVN, although at variable levels. After in vitro stimulation with the respective peptides, CTLs directed against VP1(p44) were more abundant than against VP1(p108) in most healthy individuals, while the converse was true in KTx recipients with PVN, suggesting a shift in epitope immunodominance in the setting of active BKV infection. A strong CTL response in KTx recipients with PVN appeared to be associated with decreased BK viral load in blood and urine and low anti-BKV antibody titers, while a low or undetectable CTL response correlated with viral persistence and high anti-BKV antibody titers. These results suggest that this cellular immune response is present in most BKV-seropositive healthy individuals and plays an important role in the containment of BKV in KTx recipients with PVN. Interestingly, the BKV CTL epitopes bear striking homology with the recently described CTL epitopes of the other human polyomavirus JC (JCV), JCV VP1(p36) and VP1(p100). A high degree of epitope cross-recognition was present between BKV and corresponding JCV-specific CTLs, which indicates that the same population of cells is functionally effective against these two closely related viruses.     

Genotyping of Polyomavirus BK by Real Time PCR for VP1 Gene

Polyomavirus BK latently persist in different sites, including the renourinary tract, and may reactivate causing nephropathy in renal transplant recipients or hemorrhagic cystitis in bone marrow recipients. Based on the sequence of the VP1 gene, four genotypes have been described, corresponding to the four serologically differentiated subtypes I–IV, with different prevalence and geographic distribution. In this study, the development and clinical validation of four different Real-Time PCR assays for the detection and discrimination of BKV genotypes as a substitute of DNA sequencing are described. 379 BK VP1 sequences, belonging to the main four genotypes, were aligned and “hot spots” of mutation specific for all the strains or isolates were identified. Specific primers and probes for the detection and discrimination of each genotype by four Real-Time PCR assays were designed and technically validated. Subsequently, the four Real-Time PCR assays were used to test 20 BK-positive urine specimens from renal transplant patients, and evidenced a prevalence of BK genotype I, as previously reported in Europe. Results were confirmed by sequencing. The availability of a rapid and simple genotyping method could be useful for the evaluation of BK genotypes prevalence and studies on the impact of the infecting genotype on viral biological behavior, pathogenic role, and immune evasion strategies.     

Involvement of cytoskeletal components in BK virus infectious entry

Posttransplant reactivation of BK virus (BKV) in the renal allograft progresses to polyomavirus-associated nephropathy in 1% to 8% of kidney recipients. Graft dysfunction and loss in 30% to 45% of polyomavirus-associated nephropathy-affected patients are secondary to extensive tubular epithelial cell injury induced by the lytic replication of BKV. The early events in productive BKV infection are not thoroughly understood. We have previously shown that BKV enters cells by caveola-mediated endocytosis. In this report we examine the role of microfilaments and microtubules during early viral infection. Our results show that BKV infection of Vero cells is sensitive to nocodazole-induced disassembly of the microtubule network for the initial 8 hours following virus binding. In contrast, suppression of microtubule turnover with the stabilizing agent paclitaxel has no effect on BKV infectivity. Selective disassembly of the actin filaments with latrunculin A does not impede BKV infection, while inhibition of microfilament dynamics with jasplakinolide results in reduced numbers of viral antigen-positive cells. These data demonstrate that BKV, like other polyomaviruses, relies on an intact microtubule network during early infection. BKV, however, does not share the requirement with the closely related JC virus for an intact actin cytoskeleton during intracellular transport.     

The role of sialic acid in human polyomavirus infections

JC virus (JCV) and BK virus (BKV) are human polyomaviruses that infect approximately 85% of the population worldwide [1,2]. JCV is the underlying cause of the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML), a condition resulting from JCV induced lytic destruction of myelin producing oligodendrocytes in the brain [3]. BKV infection of kidneys in renal transplant recipients results in a gradual loss of graft function known as polyomavirus associated nephropathy (PVN) [4]. Following the identification of these viruses as the etiological agents of disease, there has been greater interest in understanding the basic biology of these human pathogens [5,6]. Recent advances in the field have shown that viral entry of both JCV and BKV is dependent on the ability to interact with sialic acid. This review focuses on what is known about the human polyomaviruses and the role that sialic acid plays in determining viral tropism.     

Periodic assessment of urine and serum by cytology and molecular biology as a diagnostic tool for BK virus nephropathy in renal transplant patients

OBJECTIVE: To investigate the significance of polyomavirus (PV) viruria and viremia by morphologic, immunohistochemical and molecular analysis (multiplex nested-polymerase chain reaction) in renal transplant patients. STUDY DESIGN: Urine (n=328), serum (n= 53) and renal biopsies (n=24) from renal transplant patients (n=106) were studied. RESULTS: Decoy cells were found in 53 samples (16%) from 19 patients (18%); viral DNA was amplified in all urinary samples and disclosed BK virus (BKV) (n=24), JC virus (JCV) (n=16), and JCV and BKV DNA (n=13). BKV was the prevailing genotype in patients with a high frequency of decoy cell excretion (p = 0.001). JCV excretion correlated with a low number (p = 0.01) and BKV with a high number of decoy cells (p=0.003). PV DNA was amplified from 30/53 serum samples (56.6%); BKV was the prevailing genotype (p = 0.04). On 24 renal biopsies (18 from the decoy cell-negative and 6 from the decoy cell-positive group) PV nephropathy (PVN) was identified and BKV DNA amplified in 4 biopsies, all from the group with a high frequency of decoy cell excretion. PVN was not identified in renal biopsies from the decoy cell-negative group. CONCLUSION: PV infection is frequent in renal transplant patients. The BKV genotype in urine and serum is significantly related to a high frequency and high number of decoy cells. PVN occurs only in patients with BKV viremia and a high number and frequency of decoy cell excretion in urine. In the absence of decoy cells, PVN can be excluded. Cytologic analysis of urine is an important diagnostic tool for screening renal transplant patients at risk of PVN.     

Quantitative viral load measurement for BKV infection in renal transplant recipients as a predictive tool for BKVAN

Infection by polyomavirus BK (BKV) is an emerging problem in the clinical management of renal transplant patients because it is responsible for nephropathy and consequently can cause loss of the transplanted organ (BKV associated nephropathy, BKVAN). Aim of this study was to evaluate the use of blood viral load measurement as a screening tool for diagnosis of BKV infection and to identify a threshold value for the management of patients. A total of 75 kidney transplant patients, corresponding to 338 consecutive plasma samples, were analyzed by an automatic system for nucleic acid extraction and quantitative real-time polymerase chain reaction (PCR) for detection of BKV. BKV was detected in 170 samples (26 patients) with a median viral load of 4.1 log10 copies/mL; among these 26 patients, seven (34.7%) were found to have BKVAN on allograft biopsy together with a median viral load of 5 log10 copies/mL. The ROC curve analysis identified a viral load equal to 4.1 log10 copies/mL as the best discriminant cut-off value to predict the disease and to identify patients at risk of developing BKVAN.     

The kinetics of urinary shedding of BK virus in children with renal disease

Children with renal diseases are typically treated with immunosuppressive drugs, which place them at high risk of reactivation of the BK virus (BKV). Currently, little is known about the impact of immunosuppressive drugs on the kinetics of urinary shedding of BKV and viral reactivation in pediatric patients with renal diseases. Urine samples were collected monthly for 1 year from 20 children (median age, 9 years; range, 4–15 years) with renal diseases and subjected to real‐time PCR. Urinary shedding of BKV was detected in 35% (7/20) of the patients, three of these patients having persistent viral DNA excretion (two cases, twelve times; one case, four times) and four having intermittent viral DNA excretion. Thirty‐four of the 240 urine samples contained BKV DNA (median copy numbers, 5.66 log copies/mL; range, 2.45–7.69 log copies/mL). In two of the cases with persistent viral shedding, high copy numbers (range, 4.57–7.69 log copies/mL) of BKV DNA were detected in all 12 urine samples collected. In the other case with persistent viral excretion, a range of 2.45–3.98 log copies/mL of BKV DNA was detected in the four urine samples collected between the 9th and 12th sampling time points. Additionally, high copy numbers (range, 3.12–4.36 log copies/mL) of BKV DNA were detected intermittently in the urine samples of the other four cases. No remarkable correlations were found between the kinetics of BKV DNA loads and urinary findings such as proteinuria and hematuria. The present data demonstrate the kinetics of urinary BKV shedding in pediatric patients with renal diseases. Additionally, no pathogenic role for BKV infection was identified.