title: Genotyping of polyomavirus BK by Real Time PCR for VP1 gene.
abstract: 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.
authors.affiliation: Virology Unit, University Hospital San Giovanni Battista di Torino, Via Santena 9 10126, Turin, Italy. stefano.gambarino@unito.it
authors.forename: Stefano
authors.initials: S
authors.lastname: Gambarino
authors.forename: Cristina
authors.initials: C
authors.lastname: Costa
authors.forename: Sara
authors.initials: S
authors.lastname: Astegiano
authors.forename: Elsa Alessio
authors.initials: EA
authors.lastname: Piasentin
authors.forename: Giuseppe P
authors.initials: GP
authors.lastname: Segoloni
authors.forename: Rossana
authors.initials: R
authors.lastname: Cavallo
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Bergallo
mesh_terms: D001739:BK Virus / Q000145:classification / Q000235:genetics; D001483:Base Sequence; D036022:Capsid Proteins / Q000235:genetics*; D017931:DNA Primers; D004279:DNA, Viral / Q000737:chemistry / Q000652:urine; D005838:Genotype; D006801:Humans; D016030:Kidney Transplantation; D008969:Molecular Sequence Data; D027601:Polyomavirus Infections / Q000821:virology; D015995:Prevalence; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*; D014412:Tumor Virus Infections / Q000821:virology; D019562:Viral Load
chemical_list: D036022:Capsid Proteins; D017931:DNA Primers; D004279:DNA, Viral; C062481:VP1 protein, polyomavirus