BK Virus Subtypes Research Articles

Molecular characterization of BK virus detected in renal transplant patients in Sri Lanka: a preliminary study.

BK virus (BKV) is a polyomavirus and cause of a common infection after renal transplantation which could be preceded to BKV-associated nephropathy. It has four main subtypes (I-IV). BKV subtypes II and III are rare, whereas subtype I shows a ubiquitous distribution. The objective of the present study was to investigate the prevailing BKV subtypes and subgroups in renal transplant patients in Sri Lanka. The presence of BKV in urine was tested through virus load quantification by real-time PCR from 227 renal transplant patients who were suspected to have BKV infection. Of these patients only 41 were found to be BKV infected (>103 copies/ml) and those were subjected to conventional PCR amplification of VP1 gene followed by BKV genotyping via phylogenetic analysis based on DNA sequencing data. Persistent BK viral loads varied from 1×103 to 3×108 copies/ml. Of the 41 patient samples, 25 gave positive results for PCR amplification of subtyping region of VP1 gene of BKV. BKV genotyping resulted in detecting subtype I in 18 (72%) and subtype II in seven (28%) patients. BKV subgroups of Ia, Ib-1 and Ib-11, and Ic were identified with frequencies of 6/18 (33.3%), 6/18 (33.3%), 5/18 (27.8%), and 1/18 (5.6%), respectively. Findings from this preliminary study showed a high occurrence of subtype I, while the presence of subtype II, which is rare and less prevalent, was a novel finding for this Asian region. This emphasizes the need for further molecular and serological studies to determine the prevalence of different BKV subtypes in Sri Lanka.

Human Memory B Cells Harbor Diverse Cross-Neutralizing Antibodies against BK and JC Polyomaviruses

Human polyomaviruses cause a common childhood infection worldwide and typically elicit a neutralizing antibody and cellular immune response, while establishing a dormant infection in the kidney with minimal clinical manifestations. However, viral reactivation can cause severe pathology in immunocompromised individuals. We developed a high-throughput, functional antibody screen to examine the humoral response to BK polyomavirus. This approach enabled the isolation of antibodies from all peripheral B cell subsets and revealed the anti-BK virus antibody repertoire as clonally complex with respect to immunoglobulin sequences and isotypes (both IgM and IgG), including a high frequency of monoclonal antibodies that broadly neutralize BK virus subtypes and the related JC polyomavirus. Cryo-electron microscopy of a broadly neutralizing IgG single-chain variable fragment complexed with BK virus-like particles revealed the quaternary nature of a conserved viral epitope at the junction between capsid pentamers. These features unravel a potent modality for inhibiting polyomavirus infection in kidney transplant recipients and other immunocompromised patients.

Comparative evaluation of laboratory developed real-time PCR assays and RealStar® BKV PCR Kit for quantitative detection of BK polyomavirus

BackgroundQuantitative, viral load monitoring for BK virus (BKV) by real-time PCR is an important tool in the management of polyomavirus associated nephropathy in renal transplant patients. However, variability in PCR results has been reported because of polymorphisms in viral genes among different subtypes of BKV, and lack of standardization of the PCR assays among different laboratories. In this study we have compared the performance of several laboratory developed PCR assays that target highly conserved regions of BKV genome with a commercially available, RealStar® BKV PCR Kit. MethodThree real-time PCR assays (i) VP1 assay: selected from the literature that targets the major capsid protein (VP1) gene (ii) VP1MOD assay: VP1 assay with a modified probe, and (iii) BKLTA assay: newly designed assay that targets the large T antigen gene were assessed in parallel, using controls and clinical specimens that were previously tested using RealStar® BKV PCR Kit (Altona Diagnostics GmbH, Hamburg, Germany). Nucleic acid from all samples were extracted using the QIA symphony virus/bacteria kit on an automated DNA extraction platform QIA symphony SP (Qiagen). Primer and probe concentration, and reaction conditions for laboratory developed assays were optimized and the limit of detection of different assays was determined. Positive control for laboratory developed BK assays was prepared through construction of a plasmid carrying respective amplicon sequences. ResultsThe 95% detection limit of VP1, VP1MOD and BKLTA assays were 1.8×102, 3×103 and 3.5×102 genomic copies/ml, respectively, as determined by Probit regression analysis of data obtained by testing a dilution series of a titered patient specimen, using RealStar® BKV PCR Kit. The inter-assay and intra-assay, coefficient of variations of these assays using calibrated, plasmid standards were <1%. All assays, including the RealStar® BKV PCR assay, were highly specific when tested against a panel of external proficiency specimens containing both BK and JC viruses. All assays, except the VP1MOD assay determined BK viral load in proficiency specimens within the same log values. With reference to results obtained by RealStar® BKV PCR assay, the sensitivity and specificity of different assays tested in 116 serum specimens submitted for BK viral load assay were 91% and 97% for VP1 assay, 88% and 97% for VP1MOD assay, and 97% and 98% for BKLTA assay, respectively. BK Viral load in positive specimens determined by various assays was highly correlated (R2>0.97), based on linear regression analysis. ConclusionsThe performance characteristics of the newly designed, BKLTA assay were highly comparable to RealStar® BKV PCR assay, and can be used for routine detection and viral load monitoring of BKV in a cost-effective manner.

Viral Origin, Clinical Course, and Renal Outcomes in Patients With BK Virus Infection After Living-Donor Renal Transplantation.

BK virus (BKV) nephropathy remains the main cause of renal graft loss after living-donor renal transplantation. The aim of the study was to investigate the source and factors influencing the course of BKV infection. We investigated 214 living donor-recipient pairs. Urine and blood of donors and recipients were tested by qPCR for the presence of BKV DNA before and after transplantation; genotyping of BKV subtypes was performed. Eighty-five recipients (40%) had posttransplant BK viruria including 61 with additional viremia and 22 with nephropathy. Pretransplant urinary BKV shedding of donor or recipient was a significant risk factor for posttransplant viruria and viremia (OR, 4.52; CI, 2.33-8.77; P < 0.0001) and nephropathy (OR, 3.03; CI, 1.16-7.9; P = 0.02). In the BKV nephropathy group, urine and blood became BKV positive earlier than in the group with viruria and viremia. Renal function was worse in BKV-nephropathy compared with BKV-negative patients beginning at transplantation. Comparing BKV subtypes of donor and recipient before with the subtype of the infected recipient after transplantation, donor-derived transmission was identified in 24 of 28 corresponding pairs. BKV subtype IV had a higher prevalence in recipients with BKV nephropathy than in those with viruria and viremia (P = 0.045). Pretransplant urinary BKV shedding of donor and recipient is a risk for posttransplant infection. Donor-derived BKV transmission is an important mode of infection. BKV subtype IV may be one of the viral determinants. Early BKV positivity of urine and blood indicates later BKV nephropathy. Decreased renal function may favor BKV infection.

Frequency and subtype of BK virus infection in Iranian patients infected with HIV.

Human polyomavirus BK virus (BKV) is a double-stranded DNA virus that infects approximately 90 % of the general population as a subclinical or mild infection. In immunosuppressed patients, such as HIV cases, BKV may be reactivated resulting hemorrhagic cystitis and tubulointerstitial nephritis. However, there are limited studies on prevalence and molecular epidemiology of BKV in Iran. We therefore aimed to evaluate the prevalence and subtypes of BKV in Iranian HIV patients. A total of 99 patients with HIV infection were enrolled in the study. Presence of BKV DNA in plasma was evaluated by nested PCR. PCR products were sequenced directly, and phylogenetic analysis was performed. BKV DNA was detected in 8.08 % of HIV patients. BKV viremia presented in 4 out of 25 patients (16 %) not receiving antiretroviral therapy in comparison with 4 out 74 of HAART-treated patients (5.4 %) (P = 0.023). In patients with CD4 counts ≥200 cells/mm(3), viremia was found more commonly (7/80 = 8.8 %) than in those with lower counts (1/19 = 5.2 %) (not significant). All sequenced BKV isolates belonged to subtype Ib-2. Our findings indicated that the prevalence of BKV viremia is relatively prevalent in patients with HIV infection and significantly higher in naïve than HAART-treated cases. Therefore, HAART can eliminate BKV infection from plasma and reduce viremia although the actual implication of BKV viremia in HIV patients is not clear.

Prevalence and Distribution of BK virus Subtypes in Renal Transplant Recipients Referred to Golestan Hospital in Ahvaz, Iran.

Background:BK virus (BKV) belongs to the human Polyomaviridae and the primary BKV infection is occurred during childhood then the virus could be latent through life, especially in the kidneys and urinary system. It became reactive after an immunocompromised status, such as pregnancy or transplantation. Isolated BKV from different locations of the world is grouped into four subtypes using serological and genotyping methods. The BKV subtype I is the dominant one and has worldwide distribution.Objectives:According to our knowledge, there are no data about the BKV prevalence and its genotypes in southwest part of Iran. Considering the high prevalence of renal failure and kidney transplant patients in this part, and the role of BKV in graft rejection, this study aimed to determine the prevalence of BKV infection in renal transplant recipients referred to Golestan Hospital in Ahvaz City, Iran.Patients and Methods:Urine samples were collected from 122 kidney transplant recipients referred to Golestan Hospital in Ahvaz, southwest of Iran. The extracted DNA was amplified by Polymerase Chain Reaction, and subtype of each positive sample was determined using Restriction Fragment Length Polymorphism (RFLP) and sequencing methods.Results;From all study population, 51/122 (41.8%) urine samples were positive for BKV DNA and the other samples were negative (71/122). Forty-eight cases (94.11%) were subtype I and 3 others (5.89%) were subtype IV using the RFLP method. None of the patient’s urine samples were positive for subtypes II and III.Conclusions:Our work is the second study in Iran and considering huge numbers of transplantation in Iran and Khuzestan Province, south western of Iran, in addition to the role of this virus in kidney transplant rejection, routine evaluation of BKV positivity is recommended both for graft recipient and donors. This helps better transplantation result and may prevent graft rejection.

BK Virus Subtype III Is Associated with Hemorrhagic Cystitis After Allogeneic Hematopoietic Stem Cell Transplantation

BK Virus Subtype III Is Associated with Hemorrhagic Cystitis After Allogeneic Hematopoietic Stem Cell Transplantation

Prevalence and subtypes of BK virus in pediatric renal transplant recipients in Russia

BKV reactivation is associated with impaired graft function in kidney transplant patients. The objective of our study was to determine the prevalence of BKV infection in consecutive pediatric kidney transplant recipients at our center. Fifty-eight pediatric kidney transplant recipients were studied. The mean age at screening was 9.4 ± 2.8 yr, and samples were obtained at a median of 2.4 ± 1.4 yr after transplantation. BKV-DNA was analyzed in urine and plasma by quantitative PCR. Occurrences of BK-DNAuria and BK-DNAemia did not change in the first two yr after transplantation in children and amounted to 21-23% and 7-8%, respectively (p > 0.05). In the third year, the occurrences of BK-DNAuria and BK-DNAemia increased insignificantly to 27% and 9% in the pediatric patients. We also determined the subtypes and subgroups of BK virus isolated from Russian renal transplant recipients and found that BKV isolates were composed of subtypes Ib-2 and IV/c2. The data we obtained indicate that although only 5% of BKVAN cases occurred between years two and five post-transplantation, it seems necessary to regularly monitor pediatric patients for BKV infection through the third year after transplantation.

Rare subtypes of BK virus are viable and frequently detected in renal transplant recipients with BK virus-associated nephropathy

BK virus-associated nephropathy (BKVN) occurs in up to 5% of kidney transplants and is a significant cause of graft loss. Four major subtypes of BKV have been described, with the vast majority of individuals persistently infected with BKV Type I (> 80% of the population). Sequencing of BKV isolates subcloned from BKVN patients revealed a high percentage of variants in the urine (40%) in the VP1 subtyping region. In vitro analysis of several viral variants revealed that all variants recovered from the urine of BKVN patients produced infectious viral particles and were replication competent in cell culture while some of the variants induced cytopathic changes in infected cells when compared to the major BKV subtype, VP1 Type I. These results suggest that rare BKV VP1 variants are more frequently associated with disease and that some variants could be more cytopathic than others in renal transplant recipients.

Polymorphisms of the BK virus subtypes and their influence on viral in vitro growth efficiency

The major capsid protein, VP1, of the human polyomavirus BK (BKV) is structurally divided into five outer loops, referred to as BC, DE, EF, GH, and HI. The BC loop includes a short region, named the BKV subtyping region, spanning nucleotides 1744–1812 and characterized by non-synonymous nucleotide polymorphisms that have been used to classify different strains of BKV into four subtypes. The aim of this study was to determine if the nucleotide changes clustered within the BKV subtyping region may influence the in vitro growth efficiency of the virus. We therefore infected the African Green Monkey kidney cell line Vero with four different viral strains (named BKV I, II, III, and IV) that contained the nucleotide sequences of the BKV subtypes within the same genomic background. Infected cells were followed for 59 days and viral replication was assessed at different time points by quantitative real-time PCR (Q-PCR). BKV I, II, and IV were successfully propagated over time in Vero cells, whereas BKV III viral loads progressively decreased during the infection course, demonstrating that the non-synonymous nucleotide polymorphisms of subtype III confer a strong disadvantage for viral replication. Since subtype III differs from all the other subtypes at position 68 of the VP1, where Leu is replaced by Gln, we created viral strains bearing Gln at this position together with the polymorphisms of subtypes I, II, IV and tested their growth in Vero cells. Our results demonstrate that this amino acid substitution does not lower the replication efficiency of subtypes I, II, and IV. In conclusion, this study provides further insights to the importance of the BC loop of BKV in the virus life cycle. In addition, given the effect of the amino acid substitutions of the four BKV subtypes on infectious spread of the virus, our results suggest the need to investigate their potential association with BKV related complications.

Viral infection in bone marrow transplants: Is JC virus involved?

Hemorrhagic cystitis is characterized by hematuria due to inflammation of the bladder. In bone marrow transplants, this disease is linked to the infection by human polyomavirus BK, whereas the role of the human polyomavirus JC is unclear. The transcriptional control regions of both viruses contain important cellular transcription factor binding sites that undergo rearrangement process generating suitable variants that could be more active for viral replication and for the onset of hemorrhagic cystitis. In this study urine obtained from seven patients with bone marrow transplant were examined. Polyomavirus genomes were quantified by PCR and viral loads were compared. The transcriptional regions of both viruses were amplified and sequenced to determine the presence of variants. Subtypes of polyomaviruses were determined by amplification and sequencing of the viral protein 1 region. The results showed that four of seven patients were positive for BK DNA, two of seven patients had BK and JC DNA and one of seven had JC DNA. Positive samples were amplified and sequenced successively for transcriptional regions. The viral archetype was always found in both viruses. Finally, typing showed that BK virus subtype I infected patients with BK, whereas JC virus genotype IA and genotype 1B were found in patients infected with JC. The data suggest that new and different approaches are required to improve the morbidity and mortality caused by polyoma-associated hemorrhagic cystitis, since it known that BK virus is involved in the onset of hemorrhagic cystitis, whereas the role of JC virus should be investigated further.

Evolution of four BK virus subtypes

BK viruses (BKV) comprise four subtypes that are distinguishable by serological and molecular methods with the latter indicating up to four subgroups within subtype I. In this study, the phylogeny of all BKV subtypes was analyzed. Phylogenetic analyses of the viral structural protein VP1, concatenated sequences including the T-Ag, t-Ag, VP1 and VP2 genes and the entire coding region of BKV, each employing several tree inference methods, consistently revealed seven strongly supported clades that correlate with BKV subtypes and subgroups. In general, subtype II and III viruses were found to be sister groups, the genetic distances between which were significantly lower than those between either and the other BKV subtypes. Two benchmarks of human evolution (emergence of modern humans, 200,000 years ago; out-of-Africa migration, 100,000 years ago) were assumed for the internal calibration of BKV evolution. Utilization of either calibration point resulted in the diversification of most BKV subgroups coinciding with human radiation less than 50,000 years ago. Another approach, external calibration by linking BKV divergence to the evolution of mammals, was rejected as it advances BKV divergence prior to the emergence of modern humans.

The non-coding region of BK subtype II viruses

Four BK virus (BKV) subtypes are distinguishable by serological and molecular methods. The type I followed by type IV represents the most common subtype. Subtypes II and III occur rarely. The complete sequence of a German subtype II isolate was determined. The lack of data on the structure of the non-coding region (NCR) of subtype II and III viruses prompted us to study this part of the genome in more detail. Sequence comparisons revealed that the archetypal configuration of subtype I strain WW seems to be predominant among BKV subtypes.

BK virus subtype IV nephropathy occurring 5 years after kidney transplantation

We report on a BK virus-associated nephropathy in a 28-year-old man. His symptoms occurred 5 years after he had received a kidney transplantation. He was treated with tacrolimus, azathioprine, and prednisolone. The progress of the disease was monitored by quantitative real-time polymerase chain reactions for BK virus DNA. An analysis of viral DNA showed that the BK virus in the patient's plasma belonged to genotype IV.

Evolution of BK Virus Based on Complete Genome Data

The human polyomavirus BK virus (BKV) is ubiquitous in humans, infecting children asymptomatically. BKV is the only primate polyomavirus that has subtypes (I-IV) distinguishable by immunological reactivity. Nucleotide (nt) variations in a major capsid protein (VP1) gene region (designated the epitope region), probably responsible for antigenic diversity, have been used to classify BKV isolates into subtypes. Here, with all the protein-encoding gene sequences, we attempted to elucidate the evolutionary relationships among 28 BKV isolates belonging to subtypes I, III, and IV (no isolate belonging to subtype II, a minor one, was included). First, using the GTR + Gamma + I model, maximum likelihood trees were reconstructed for individual viral genes as well as for concatenated viral genes. On the resultant trees, the 28 BKV isolates were consistently divided into three clades corresponding to subtypes I, III, and IV, although bootstrap probabilities are not always high. Then we used more sophisticated likelihood models, one of which takes account of codon structure, to elucidate the phylogenetic relationships among BKV subtypes, but the phylogeny of the deep branchings remained ambiguous. Furthermore, the possibility of positive selection in the evolution of BKV was examined using the nonsynonymous/synonymous rate ratio as a measure of selection. An analysis based on entire genes could not detect any strong evidence for positive selection, but that based on the epitope region identified a few sites potentially under positive selection (these sites were among those showing subtype linked polymorphisms).

Unique BK virus non-coding control region (NCCR) variants in hematopoietic stem cell transplant recipients with and without hemorrhagic cystitis

Hematopoietic stem cell transplant recipients frequently develop BK virus (BKV)-associated hemorrhagic cystitis, which coincides with BK viruria. However, the precise role of BKV in the etiology of hemorrhagic cystitis in hematopoietic stem cell transplant recipients remains unclear, since approximately 50% of all such adult transplant recipients excrete BKV, yet do not develop this clinical condition. In the present study, BKV were analyzed to determine if mutations in the non-coding control region (NCCR), and specific BKV sub-types defined by sequence analysis of major capsid protein VP1, were associated with development of hemorrhagic cystitis in hematopoietic stem cell transplant recipients. The regions encoding VP1 and NCCRs of BKV in urine samples collected from 15 hematopoietic stem cell transplant recipients with hemorrhagic cystitis and 20 without this illness were amplified and sequenced. Sequence variations in the NCCRs of BKV were identified in urine samples from those with and without hemorrhagic cystitis. Furthermore, five unique sequence variations within transcription factor binding sites in the canonical NCCR, O-P-Q-R-S, were identified, representing new BKV variants from a population of cloned quasi-species obtained from patients with and without hemorrhagic cystitis. Thirty-five BKV VP1 sequences were analyzed by phylogenetic analysis but no specific BKV sub-type was associated with hemorrhagic cystitis. Five previously unrecognized naturally occurring variants of the BKV are described which involve amplifications, deletions, and rearrangements of the archetypal BKV NCCRs in individuals with and without hemorrhagic cystitis. Architectural rearrangements in the NCCRs of BKV did not appear to be a prerequisite for development of hemorrhagic cystitis in hematopoietic stem cell transplant recipients.

Prevalence of BK virus subtype I in Germany

The primary infection with human polyomavirus BK (BKV) occurs in early childhood and leads to viral latency within the urogenital tract. Up to 90% of the adult population are seropositive. In immunosuppressed patients, the BKV may be reactivated resulting in typical disease patterns like hemorrhagic cystitis and tubulointerstitial nephritis. Based on serological and molecular methods, BKV isolates were classified into four subtypes previously. Sixty specimens obtained from German renal and bone marrow transplant recipients were analyzed to gain data on the prevalence of BKV subtypes in Germany. With 90.9%, BKV subtype I was found to be predominant in both patient groups. 6.1% of BKV strains were classified as subtype IV. This pattern of phylogenetic distribution is similar to that demonstrated previously in England, Tanzania, the United States and Japan. Remarkably, there was one German BKV virus with a sequence which clusters together with strain SB in subtype II. The BKV subtype I was found to consist of at least three subgroups designated as Ia, Ib, and Ic. While the majority of the German sequences represent subgroup Ic, most of the Japanese sequences are clearly distinct. These findings support the hypothesis of distinct geographical prevalence of BKV subgroups. For the genotyping region, a relationship of BKV subgroups to disease patterns like hemorrhagic cystitis or tubulointerstitial nephritis could not be demonstrated.

BK virus subtype 1 infection associated with tubulointerstitial nephritis in a renal allograft recipient

The BK polyomavirus (BKV) infects most of the human population, but clinically relevant infections are usually limited to individuals who are in an immunosuppressed state. The significance of BKV infection was investigated in a 50-year-old man who underwent cadaveric kidney transplantation and was treated with tacrolimus, mycophenolate mofetil and prednisolone. By staining renal biopsy specimens with a monoclonal antibody against BK large T antigen, we were able to observe the relationship between the appearance of the BKV antigen and the extent of immunosuppression in this patient. We also determined that BKV belonged to genotype I by analysis of viral DNA from the patient's urine.

Detection of BK polyomavirus genotypes in healthy and HIV-positive children.

Urine samples from 211 community children (3-7 years age), from 33 HIV type-1 infected children and from 56 HIV-negative children were collected and analyzed for the presence of BK virus (BKV) DNA by PCR. PCR amplifications were carried out using primers specific for the BKV structural region VP1. We also investigated the distribution of BKV subtypes by a restriction fragment polymorphism assay (RFLP). We demonstrated BKV DNA in 3.8% of 211 community children with a higher prevalence of subtype I. In HIV-1 positive children we detected BKV DNA in 2 urine samples (6%) out of 33, both belonging to subtype I. The HIV-negative cluster did not show any positivity to BKV DNA. The results confirm a more frequent primary BKV infection in children of 3-5 years of age and a higher prevalence in hospitalized children affected by HIV-1. The most relevant finding was that among both the community and HIV-1 positive children the subtype I was the most frequently detected.

Prevalence and distribution of BK virus subtypes in healthy people and immunocompromised patients detected by PCR-restriction enzyme analysis

Four antigenic subtypes of BK virus (BKV) have recently been characterised by both genomic subtyping and serological reactivity. To study the prevalence and distribution of subtypes of BKV in different groups of patients. Urine specimens were collected from 33 bone marrow transplant (BMT) recipients, from 101 HIV-infected patients, from 15 children aged 2-5 and from 40 pregnant women were tested for BKV DNA by polymerase chain reaction (PCR) and subtyped using a PCR-sequencing (PCR-S) and a modified PCR-restriction enzyme analysis (PCR-RE) methods. BKV DNA was detected in 12/18 (67%) of BMT patients with haematuria and 5/15 (33%) without. Overall BKV DNA was detected in 45% of HIV-infected patients, the prevalence of BKV DNA increased with greater immunosuppression as defined by CD4 cell counts. BKV DNA was detected in urine samples from 27% of children and 47% of pregnant women. Four stable BKV subtypes were detected in these patient groups. Dual infections with more than one subtype were identified in urine samples from HIV-infected patients, children and pregnant women but not in the samples from bone marrow recipients. This study has confirmed the high prevalence of BKV infection in immunocompromised patients and suggests that stable BKV subtypes with conserved sequences are circulating in the human population. The techniques of PCR-S and PCR-RE described in this study are sufficiently sensitive for subtyping BKV direct from clinical specimens.