Phylogenetic Analysis of Polyomavirus BK Sequences

Abstract

Polyomavirus BK (BKV) has emerged as an important pathogen in kidney transplant patients. Existing taxonomic classifications of BKV come from conventional DNA sequence alignments based on limited data derived from the VP1 gene. We have used a phylogenetic whole-genome approach to examine the pattern of diversity and evolutionary relationships between 45 BKV strains isolated from multiple clinical settings. This analysis supports the classification of BKV into six genotypes, of which types V and VI have not been previously recognized. BKV strains hitherto classified as type I are, in fact, quite heterogeneous, and several cluster with our newly defined genotypes V and VI. The sequence information needed for assigning genotypes can be captured by VP1, VP2, VP3, or large T-gene sequencing. The most polymorphic coding region in the viral genome is VP1, but significant variation is also present in the large T-antigen gene, wherein polymorphisms are found in 11.39% of all nucleotide sites, 46.22% of which are cluster specific. Type-specific amino acid changes within the VP1 region are predicted to map to the BC and DE loops. The number of taxonomically informative amino acid changes in the large T antigen exceeds even that of the VP1 region. Viral strains isolated from healthy subjects and from patients with human immunodeficiency virus infection, WiskottAldrich syndrome, and vasculopathy with capillary leak syndrome formed distinct subclusters. However, within the kidney transplant population, BKV strains derived from patients with asymptomatic viruria did not show complete separation from strains associated with allograft nephropathy.Polyomavirus BK (BKV) has a worldwide seroprevalence of 60 to 80% in humans (10). Primary infection occurs in childhood (11) and leads to viral latency in the urogenital tract and mononuclear cells. Following reactivation, which mostly occurs in immunocompromised patients, the virus is excreted in the urine (5). BKV viruria in renal transplant recipients ranges between 10 to 60% and has been associated with ureteric stenosis (16) and BK viral nephropathy characterized by tubular necrosis and interstitial nephritis (32, 33). In bone marrow transplant recipients, BKV has been linked to hemorrhagic cystitis (13,26,34).Characterization of genetic diversity of BK virus has biologic as well as clinical implications. This information is needed to seek potential relationships between viral genotype and clinical disease. Sequence data are required by diagnostic virology laboratories to ensure that primers and probes being used for amplification of viral DNA can successfully detect all naturally occurring viral strains. One of the most variable regions of the viral genome is the noncoding control region (NCCR), which shows insertions, deletions, duplications, and complex rearrangements involving enhancer and/or promoter sequences (28, 29). Significant variation is also recognized in the VP1 gene, which codes for VP1, a major structural protein that comprises approximately 80% of the total viral c...

BK Polyomavirus Genotypes Represent Distinct Serotypes with Distinct Entry Tropism

“…Our findings raise the interesting question of whether different BKV genotypes or subgenotypes exhibit altered tissue tropism or different levels of virulence in vivo. Although previous studies have surveyed BKV genotypes found in nephropathic lesions (15,16,(37)(38)(39), these studies have relied on BKV-I-biased PCR primers that underestimate the abundance of BKV genotypes II, III, and IV (40). The possibility that commonly used PCR primers do not robustly detect non-genotype I BKVs (particularly in the case of individuals coinfected with multiple BKV genotypes) is consistent with our observation that BKV-II seroprevalence (Fig.…”

Genotyping Schemes for Polyomavirus BK, Using Gene-Specific Phylogenetic Trees and Single Nucleotide Polymorphism Analysis

“…Subtypes V and VI, previously defined by us on the basis of more limited sequence data, are best considered to be subgroups of subtype I and clustered with Ib2 and Ib1, respectively. Subgroups of subtype I are shown to be definite phylogenetic entities separated by bootstrap values of 100%, which are much better than those previously obtained using more limited sequence information based on the VP1 gene (33). Bootstrap values for subgroups of subtype IV ranged from 68 to 87%.…”

Comparative Evaluation of Three Nucleic Acid-Based Assays for BK Virus Quantification

“…A fragment within the T-Ag gene, from nucleotides 2993 to 3738, was amplified and sequenced only for subtyping of genotype IV samples. DNA was amplified with Hot Start PCR master mix (Biotech Rabbit), using the published primer pairs VP1-3 plus SeqB-11 and SeqB-16 plus SeqB-19 (3,14). Cycle sequencing of the purified amplicons was performed using an Applied Biosystems BigDye Terminator v1.1 kit according to the manufacturer's instructions.…”

“…With the growing importance of BKV in the management of immunocompromised patients over recent years, several manufacturers have developed commercial blood and urine BKV DNA quantification assays based on real-time PCR technology. Our knowledge of BKV genomic diversity has also improved considerably (4,10), as a large number of studies of full-genome BKV DNA have been published over the last decade (3,11). However, it is very difficult to provide clinicians with accurate data, because most methods are in-house methods and no international standards have yet been established to allow comparisons between different tests.…”

“…In bone marrow transplant recipients, BKV reactivation may result in hemorrhagic cystitis. Molecular analyses of numerous isolates have led to the classification of the BKV genus into several subtypes (Ia, Ib1, Ib2, Ic, II, III, IVa, IVb, and IVc), based on phylogenetic analyses of full-genome viral DNA sequences (3,4). The various genotypes have a specific geographic distribution in the population (5).…”