We recruited patients with PC and BPH using intentional sampling at the Department of Urology, Chiayi Christian Hospital in Taiwan from 2016 to 2020. The inclusion criteria were as follows: (1) patients diagnosed with PC or BPH; (2) tissue samples with cancerous area higher than 50%; (3) completion of the surveys by patients with or without help. The exclusion criteria were as follows: (1) patients with a history of other cancers; (2) severe communication problems; (3) patients with missing clinically important pathological data; and (4) patients who were immunocompromised or had post-transplanted organs. All patients provided written informed consent, and their confidentiality was safeguarded. Specimens were collected after review, and the study was approved by the institutional review board of Chiayi Christian Hospital.
The clinical data of the patients in this study were used to investigate the relationship between JCPyV and BKPyV with PC and BPH in Taiwan. The patient clinical-pathological information of the patients including age, sex, tumor size, tumor tissue types, tumor-node-metastasis (TNM) stage, prostate-specific antigen (PSA) concentration, Gleason score of the patients with PC, and the age, sex, and PSA concentration of patients with BPH were collected. All patients with PC were characterized with clinical-pathological stages between Tx and T4, as defined in the American Joint Committee on Cancer (AJCC) TNM staging system for PC . A total of 76 prostate adenocarcinoma tissues and 30 BPH tissue specimens were collected, thoroughly cleaned, and processed independently for analysis. PC specimens were obtained using transrectal ultrasound biopsy, transurethral biopsy, and radical prostatectomy. BPH specimens were obtained via transurethral resection of the prostate for bladder outlet obstruction. The specimens were stored in the pathology department until analysis. Pathological tissue sections were used to investigate the presence of the JCPyV and BKPyV genomes and the expression of the viral proteins LT and VP1. A formalin-fixed, paraffin-embedded JCPyV-bearing cell line (JCI) [29, 30] was used as a positive control for PCR and immunohistochemical staining.
DNA samples were extracted from paraffin-embedded tissue using the Gene JET FFPE DNA purification reagent (Thermo Fisher, Vilnius, Lithuania) according to the instructions of the manufacturer. Briefly, tissue was deparaffinized using xylene, washed 2–3 times with absolute ethanol, washed with deionized water to remove residual ethanol, and air-dried. The sample was treated with 50 μg/ml proteinase K (Thermo Fisher, Vilnius, Lithuania) for 16–18 h at 50 °C, placed in boiling water for 10 min to deactivate proteinase K, and centrifuged at 11,180×g for 3 min. The supernatant was collected for DNA purification. DNA (200 ng) was collected for viral genome analysis using PCR . The human β-actin gene was used as an internal control to ensure successful DNA extraction from paraffin-embedded tissues and to exclude false negatives.
Nested PCR and DNA sequencing
Nested PCR was used to detect the presence of viral DNA in PC and BPH specimens. Typically, after polyomavirus infection, the genome may undergo rearrangement within regulatory regions to produce new variants . Therefore, we used two primer pairs to amplify the constant regulatory regions of JCPyV and BKPyV . The first PCR used the primer pair JBR1 and JBR2 (nucleotides 5067–5091 of the JCPyV TW-3 strain, 5′-CCTCCACGCCCTTACTACTTCTGAG-3′ and 279–255, 5′-GTGACAGCTGGCGAAGAACCATGGC-3′, respectively) to amplify the regulatory region. Next, 5 μL of the first PCR product was used as the template, and the primer pair JBRNS and JBRNAS (nucleotides 5100–5 of the JCPyV TW-3 strain, 5′-GAGGCGGCCTCGGCCTC-3′ and 227–212, 5′-GGCTCGCAAAACATGT-3′, respectively) were used for the second PCR. PCR amplification of the human beta-globin (β-globin) 5′ UTR region was performed simultaneously to ensure the presence of tissue DNA and exclude false negative results. All specimens were analyzed in triplicate. The secondary PCR products were analyzed via electrophoresis using 2.5% agarose gels. The JCPyV CY (GenBank Accession No. AB03849), and BKPyV UT (GenBank accession No. M 34049.1) genotypes were used as positive controls. The nested PCR products had a size of 243 bp and 289 bp, respectively, which were consistent with those of the regulatory regions of JCPyV and BKPyV. The PCR products were sent to Mingxin Biotechnology (Taipei City, Taiwan) for DNA sequencing analysis after purification. All sequences were compared with the JCPyV archetype (GenBank accession No. AB03849), and BKPyV prototype (GenBank accession No. M 34049.1) to confirm whether the regulatory regions underwent rearrangement and to determine the genotypes.
IHC staining was performed as previously reported  with minor modifications. Briefly, 3 μm thick formalin-fixed, paraffin-embedded tissue sections were deparaffinized with xylene for 10 min, rehydrated through a gradient of 100, 95, 80, and 70% ethanol, and equilibrated in Tris buffer (TBS; 0.1 M Tris-HCl, pH 7.4 and 0.15 M NaCl). Antigen retrieval was performed by placing the slides in 0.01 M citric buffer (pH 6.0) and autoclaving at 121 °C for 20 min. The slides were treated with a protein-blocking agent for 10 min to reduce the non-specific binding of antibodies to the tissue and subsequently washed twice. Primary monoclonal antibodies anti-SV40 LT (mia90661; Thermo Fisher, Vilnius, Lithuania) and anti-JCPyV capsid VP1 (ab34756; Abcam, Cambridge, USA) were used to detect the expression of the early viral protein LT and the late structural protein VP1, respectively. Tissue sections were incubated overnight in a humidified chamber at 37 °C, followed by incubation after the sequential addition of biotinylated secondary antibody (PK6102, Vectastain ABC kit, Burlingame, CA, USA) and avidin-biotin complex (Vectastain ABC kit, Burlingame, CA, USA) for 1 h each. After development in the presence of diaminobenzidine (DAB) substrate (Sigma–Aldrich, St. Louis, MO, USA), sections were counterstained using hematoxylin.
Transmission electron microscopy (TEM)
Four fresh tissue samples were collected from 27 PC tissues that were positive for JCPyV DNA and VP1 protein expression. The specimens that were fixed in 2.5% glutaraldehyde were cut into cubes of approximately 1 mm3. These specimens were dehydrated and embedded in LR White Resin (Polysciences, Warrington, PA, USA). The embedded specimen was subsequently sliced into 60 nm-thick sections and attached to nickel grids. After incubation in 0.25% gelatin-containing TBST buffer for 1 h, the grids were sequentially incubated with anti-JCPyV VP1 monoclonal antibody (ab34756; Abcam, Cambridge, USA) for 2 h and with 15 nm gold-conjugated protein-G (EM.PGG15; Agar, Essex, UK) for 1 h. The immunolabeled sections were treated with 2% uranyl acetate for 20 min and 0.5% lead citrate for 10 min and observed using a JEM-100CX II transmission electron microscope (JEOL, Peabody, MA, USA) .
All statistical analyses were conducted using SPSS version 18.0, for Windows (SPSS Inc., Chicago, IL, USA). Statistical significance was set at p < 0.05. Fisher’s exact test or the Mann-Whitney U test was used to analyze the differences between the prevalence of viral DNA and LT and VP1 proteins in PC and BPH tissues. Continuous evaluations were compared between groups using the Mann-Whitney U test. Fisher’s exact test was used to compare categorical variables. The relationship between the clinical characteristics of patients and the risk of JCPyV/BKPyV infection in PC cells, the odds ratio, and 95% confidence interval were evaluated using logistic regression analysis.