In Vitro Study Evaluating the Effect of Different Immunosuppressive Agents on Human Polyomavirus BK Replication.

BACKGROUND: Human polyomavirus BK (BKPyV) is the etiologic agent of polyomavirus-associated nephropathy, a leading cause of kidney transplant dysfunction. Because of the lack of antiviral therapies, immunosuppression minimization is the recommended treatment. This strategy offers suboptimal outcomes and entails a significant risk of rejection. Our aim was to evaluate the effect of different immunosuppressive drugs (leflunomide, tacrolimus, mycophenolic acid, sirolimus, and everolimus) and their combinations in an in vitro model of BKPyV infection. METHODS: Human renal tubular epithelial cells were infected with BKPyV and treated with leflunomide, tacrolimus, mycophenolic acid, sirolimus, and everolimus, administered alone or in some combination thereof. Viral replication was assessed every 24 hours (up to 72 hours) by BKPyV-specific quantitative real-time polymerized chain reaction for the VIRAL PROTEIN 1 sequence in cell supernatants and by western blot analysis targeting the viral protein 1 and the glyceraldehyde 3-phosphate dehydrogenase on total protein lysates. Results were described as viral copies/mL and compared between treatments at any prespecified time point of the study. RESULTS: The highest inhibitory effects were observed using leflunomide or everolimus plus mycophenolic acid (mean BKPyV replication log reduction 0.28). The antiviral effect of everolimus persisted when it was used in combination with tacrolimus (mean BKPyV replication log reduction 0.27). CONCLUSIONS: Our experience confirms that everolimus has anti-BKPyV properties and prompts future research to investigate possible mechanisms of action. It also provides a rational basis for targeted clinical trials evaluating alternative immunosuppressive modification strategies.

Human endogenous retroviruses env gene expression and long terminal repeat methylation in colorectal cancer patients.

Human endogenous retroviruses (HERV) are remnants of exogenous retroviral infections, representing 8% of the human genome. Their regulation is based on the DNA methylation of promoters, the long terminal repeats (LTRs). Transcripts from HERV have been associated with cancers, but reports concerning HERV expression in colorectal cancer remain sporadic. Sixty-three patients with advanced stages of colorectal cancer were enrolled in this study. The expressions of HERV env gene, and HERV-H, -K, -R and -P LTRs and Alu, LINE-1 methylation levels, were investigated in the tumor, normal adjacent tissues, and, where possible, blood and plasmatic extracellular vesicles (EVs). Associations among HERV env expression, methylation status and clinical characteristics were evaluated. No differences were observed in HERV env gene expression levels among the clinical specimens, while Alu, LINE-1, HERV-H and -K LTRs were demethylated in the tumor compared to the normal adjacent tissues (p < 0.05).The HERV env gene was expressed in the EVs at of 54% (-H), 38% (-K), 31% (-R) patients. Association was not found between HERV env expression and LTR methylation, but significant higher expression of HERV-P and -R env was found in tumor tissues arising from the right colon. Our findings do not demonstrate significant overexpression of the studied HERV in colorectal cancer, but their association with tumor localization and specificity of the changes in DNA methylation of retroelements are shown. HERV sequences were packaged in the EVs and might be transferred from one cell to another.

Merkel Cell Polyomavirus Is Associated with Anal Infections in Men Who Have Sex with Men.

BACKGROUND: Viral infections of the anal/rectal tract of men who have sex with men (MSM) have been poorly studied. METHODS: In total, 158 swab samples (81 anal/rectal, 65 throat/oral and 12 urethral) were collected from 126 MSM. DNA was isolated and subjected to real-time PCR assays for the detection of the sexually transmitted (ST) pathogens Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasmas ssp, human papillomavirus (HPV) and six human polyomaviruses (HPyVs; JCPyV, BKPyV, Merkel cell PyV⁻MCPyV-, HPyV-6, HPyV-7 and HPyV-9). RESULTS: C. trachomatis (31/126, 24.6%) and M. genitalium (30/126, 23.8%) were the most frequently detected ST pathogens. Thirty-one/126 (24.6%) patients were positive for at least one HPyV. The significantly (p < 0.05) prevalent HPyV in the anal tract was MCPyV, which was amplified in 27/81 (33.3%) samples, followed by HPyV-6, which was amplified in 6/81 (7.4%) swabs. Coinfections with MCPyV and C. trachomatis or Mycoplasmas were found in 4/21 (19.0%) and 5/21 (23.8%) anal/rectal swabs, respectively. Three/4 MCPyV-C. trachomatis coinfected patients were symptomatic. CONCLUSIONS: Based on the high prevalence of MCPyV in the anal/rectal swabs from MSM patients and on the well-known oncogenic properties of MCPyV, sexual transmission and possible involvement of HPyVs in the pathogenesis of diseases of the anal canal should be further studied.

Characterization of an in vitro model to study the possible role of polyomavirus BK in prostate cancer.

Prostate cancer (PCa) is the most common male neoplasms in the Western world. Various risk factors may lead to carcinogenesis, including infectious agents such as polyomavirus BK (BKPyV), which infects the human renourinary tract, establishes latency, and encodes oncoproteins. Previous studies suggested that BKPyV plays a role in PCa pathogenesis. However, the unspecific tropism of BKPyV and the lack of in vitro models of BKPyV-infected prostate cells cast doubt on this hypothesis. The aim of the present study was to determine whether BKPyV could (a) infect normal and/or tumoral epithelial prostate cells and (b) affect their phenotype. Normal epithelial prostate RWPE-1 cells and PCa PC-3 cells were infected with BKPyV for 21 days. Cell proliferation, cytokine production, adhesion, invasion ability, and epithelial-to-mesenchymal transition (EMT) markers were analyzed. Our results show that (a) RWPE-1 and PC-3 cells are both infectable with BKPyV, but the outcome of the infection varies, (b) cell proliferation and TNF-α production were increased in BKPyV-infected RWPE-1, but not in PC-3 cells, (c) adhesion to matrigel and invasion abilities were elevated in BKPyV-infected RWPE-1 cells, and (d) loss of E-cadherin and expression of vimentin occurred in both uninfected and infected RWPE-1 cells. In conclusion, BKPyV may change some features of the normal prostate cells but is not needed for maintaining the transformed phenotype in the PCa cells The fact that RWPE-1 cells exhibit some phenotype modifications related to EMT represents a limit of this in vitro model.

Particulate matter exposure increases JC polyomavirus replication in the human host.

BACKGROUND: Human polyomaviruses (HPyVs) asymptomatically infect the human population during childhood and establish latency in the host. Viral reactivation and urinary excretion can occur when the immune system is impaired. Exposure to particulate air pollution, including the PM10/PM2.5 components, is a public health problem and has been linked to several disorders. Studies assessing the relationship between PM10/PM2.5 exposure and viral replication are lacking. OBJECTIVES: To investigate the relationship between HPyVs viruria and PM10/PM2.5 exposures. METHODS: Individual environmental exposure was assessed in 50 healthy adult volunteers using a chemical transport model (CTM) with a municipality resolution for daily PM10 and monitoring stations data for daily PM2.5 exposures. For each subject, a urine sample was collected, and HPyVs (JCPyV, BKPyV, MCPyV, HPyV6, HPyV7 and HPyV9) loads were determined. Zero-inflated negative binomial (ZINB) regression was used to model the count data, as it contained excessive zeros. Covariates were chosen by stepwise selection. RESULTS: HPyVs DNA was detected in 54% (median:87.6*105 copies/ml) of the urine samples. JCPyV was the prevalent (48%, (median viral load:126*105 copies/ml). Considering the load of the most frequently measured HPyVs, JCPyV, in the count-part of the ZINB model, every unitary in PM measured 2 days before urine collection (PM Day -2) was associated with an increase in JCPyV load (PM10: +4.0%, p-value = 0.002; PM2.5: +3.6%, p-value = 0.005). In the zero-part, the significant predictor was the PM10 measured 5 days before urine collection (+3%, p-value = 0.03). CONCLUSIONS: The environmental levels of PM10/PM2.5 increase the JCPyV viruria. Our findings emphasize the need for studies assessing the influence of air pollution exposure on the risk of viral reactivation.

Antibody response to polyomavirus primary infection: high seroprevalence of Merkel cell polyomavirus and lymphoid tissue involvement.

Human polyomaviruses (HPyVs) asymptomatically infect the human population establishing latency in the host, and their seroprevalence can reach 90% in healthy adults. Few studies have focused on the pediatric population, and there are no reports regarding the seroprevalence of all the newly isolated HPyVs among Italian children. Therefore, we investigated the frequency of serum antibodies against 12 PyVs in 182 immunocompetent children from Northeast Italy, by means of a multiplex antibody detection system. Additionally, secondary lymphoid tissues were collected to analyze the presence of HPyV DNA sequences using a specific real-time PCRs or PCRs. Almost 100% of subjects were seropositive for at least one PyV. Seropositivity ranged from 3% for antibodies against simian virus 40 (SV40) in children from 0 to 3 years, to 91% for antibodies against WU polyomavirus (WUPyV) and HPyV10 in children from 8 to 17 years. The mean number of PyV for which children were seropositive increased with the increasing of age: 4 standard deviations (SD) 1.8 in the 0-3-year group, 5 (SD 1.9) in the 4-7-year group, and 6 (SD 2.2) in the 8-17-year group. JC polyomavirus (JCPyV) DNA was detected in 1% of the adenoids, WUPyV in 12% of the tonsils, and 28% of the adenoids, and Merkel cell polyomavirus (MCPyV) was present in 6 and 2% of the tonsils and adenoids, respectively. Our study gives new insights on the serological evidence of exposure to PyVs during childhood, and on their possible respiratory route of transmission.

Human Polyomaviruses: The Battle of Large and Small Tumor Antigens.

About 40 years ago, the large and small tumor antigens (LT-Ag and sT-Ag) of the polyomavirus (PyVs) simian vacuolating virus 40 have been identified and characterized. To date, it is well known that all the discovered human PyVs (HPyVs) encode these 2 multifunctional and tumorigenic proteins, expressed at viral replication early stage. The 2 T-Ags are able to transform cells both in vitro and in vivo and seem to play a distinct role in the pathogenesis of some tumors in humans. In addition, they are involved in viral DNA replication, transcription, and virion assembly. This short review focuses on the structural and functional features of the HPyVs' LT-Ag and sT-Ag, with special attention to their transforming properties.

Multiplex array analysis of circulating cytokines and chemokines in natalizumab-treated patients with multiple sclerosis.

Natalizumab greatly reduces inflammatory relapses in multiple sclerosis (MS) by blocking the integrin-mediated leukocyte traffic to the brain, but less is known about its effects on the systemic immunity. We measured 48 cytokines/chemokines in sera from 19 natalizumab-treated MS patients. Serum concentrations of both anti-(IL-10, IL1ra) and pro-inflammatory (IL7, IL16) molecules decreased after 21-month treatment, without associations to unbalanced Th2/Th1cytokine ratios, clinical responses, and blood/urine replication of polyomavirus JC (JCPyV). No patient developed the JCPyV-related progressive multifocal leukoencephalopathy (PML), the major risk factor of natalizumab therapy. Our data suggest that natalizumab has marginal impact on the systemic immunity.

JC virus infection is acquired very early in life: evidence from a longitudinal serological study.

JC virus (JCV) is a widespread member of the Polyomaviridae family. Following primary infection, which occurs asymptomatically during childhood, JCV establishes latency in the host. JCV seroprevalence can reach 80 % in healthy adults, but the age of viral exposure has not been yet characterized. This study was conducted to define JCV seroprevalence in Italian infants and to estimate the date of primary infection. A JCV viral protein 1 (VP1)-GST fusion protein was used in conjunction with a homemade indirect enzyme-linked immunosorbent assay (ELISA) to test for the presence of IgG antibodies to JCV in 981 serum samples collected from 644 Italian infants of different ages (1 day to 3 years old) and in 102 breast milk samples. IgM antibody presence was also evaluated in longitudinally collected samples from 17 selected children. JCV antibody prevalence and normalized optical density (nOD) were calculated. For the longitudinal analysis, generalized estimating equation techniques and spline functions were used to estimate the possible non-linear effects of time on antibody production kinetics. JCV IgG was detected in 71.8 % of the sera. Prevalence increased over time from 46.1 % (1 month old) to 80.7 % (12 months old), 85.9 % (24 months old), and 85.5 % (36 months old). As determined by nOD, the longitudinal analysis of serum IgG amounts in children of this study (ages 1 day to 3 years old) illustrated IgG kinetic changes with statistically significant trends (p = 0.001). One-month-old children were largely negative for JCV IgM (82.4 %), and 58.8 % of children produced JCV IgM within the second and sixth months of life. JCV IgG was detected in 27.3 % of breast milk samples. JCV primary infection likely occurs before 6 months of age, and a sizeable percentage of Italian infants will become JCV seropositive within 2 years of age. This study can be used to determine the optimal age for potential future JCV vaccination in infants.

Interaction Between Human Polyomavirus BK and Hypoxia Inducible Factor-1 alpha.

BK polyomavirus (BKV) has a worldwide seroprevalence of approximately 90%. After primary infection, BKV establishes a life-long latency within the urogenital tract. The severe immunological impairment occurring in renal transplant recipients leads to BKV reactivation, which may result in polyomavirus associated nephropathy (PVAN). While the transplanted kidney is transiently unperfused, Hypoxia Inducible Factors (HIFs) mediate the cellular response to hypoxia. The α-subunit of HIF isoform 1 (HIF-1α) may interact with several viruses, but until now, there has been no information regarding the interaction between BKV and HIF-1α. The aim of this study is to investigate the possible interaction between HIF-1α and BKV and its potential effect on the pathogenesis of PVAN. Screening of 17 kidney tissue samples revealed that HIF-1α expression was 13.6-fold higher in PVAN tissues compared to control tissues. A luminometric assay in co-transfected African green monkey kidney cells (VERO) demonstrated BKV promoter activation ranging from two to sixfold (P < 0.05) when HIF-1α was over-expressed. A Chromatin ImmunoPrecipitation (ChIP) assay showed structural binding between the BKV promoter and HIF-1α. The amount of BKV DNA increased by threefold in VERO infected cells that were exposed to simulated hypoxia, compared to the cells not subjected to hypoxia. Both ex vivo and in vitro interactions between HIF-1α and BKV were observed, suggesting that HIF-1α, stabilized during transplantation, may be able to bind the BKV promoter and enhance BKV replication. Thus, hypoxia should be considered a risk factor for the development of PVAN in kidney transplant recipients.

Human polyomavirus 6 DNA in the cerebrospinal fluid of an HIV-positive patient with leukoencephalopathy.

BACKGROUND: Leukoencephalopathies in HAART-treated, HIV-positive patients include progressive multifocal leukoencephalopathy (PML), a result of lytic infection oligodendrocytes by JC polyomavirus (JCV), and another form characterized by the absence of JCV genome in cerebrospinal fluid (CSF). OBJECTIVES: To test the potential viral etiology of JCV-negative leukoencephalopathy. STUDY DESIGN: CSF was collected from 43 HIV-positive patients with MRI suggestive of leukoencephalopathies. DNA was isolated and real-time PCR assays for neurotropic viruses (Herpes Simplex Viruses 1/2, Varicella Zoster Virus, Epstein Barr Virus, Human Cytomegalovirus, Human Herpesvirus 6, JCV and HIV) were conducted. CSF from 14 non-reactive cases were subjected to random nucleic acid amplification, deep sequencing, and in silico search for viral sequences. RESULTS: JCV genome was detected in the CSF of 19/43 PML patients, HIV genome in the CSF of 5 PML patients including 2 JCV negative patients, and no viruses were detected in 22 patients. Human Polyomavirus 6 (HPyV6) DNA was detected by deep sequencing in one JCV-negative leukoencephalopathy CSF sample. CONCLUSIONS: HPyV6 DNA was detected in CSF of a case of demyelinating disease. HPyV6 has not been previously reported in CSF or associated with any disease. Demonstrating a causative role will require further studies.

Evidence supporting the association of polyomavirus BK genome with prostate cancer.

Prostate cancer (PCA) is the most frequent cancer in men. Exposure to infectious agents has been reported to have a putative role in tumorigenesis. Among the infectious agents, convincing evidence has been accumulated about the human polyomavirus BK (BKV). Tissue fresh specimens, serum, and urine samples were collected from 124 consecutive patients, 56 with PCA and 68 with benign prostatic hyperplasia (BPH). Quantitative PCR assays were used to assess the presence of BKV and JC virus (JCV) genomes. BKV-positive tissue specimens were found in 32.1 and 22.1 % of PCA and BPH patients, respectively; in PCA group the number of positive BKV specimens/patients was significantly higher than in BPH group (3.06 vs. 1.73, p = 0.02). JCV genome was found in the biopsies collected from 28.1 and 24.2 % of PCA and BPH patients, respectively, with no significant difference in the rate of JCV specimens/patients between PCA and BPH groups. Our results support the putative causal association between BKV genome and PCA. Further studies are required to demonstrate the direct pathogenetic role of BKV in the PCA occurrence and progression in order to clear the tempting way of vaccine prophylaxis.