JCV Infection Research Articles

JC virus spread is potentiated by glial replication and demyelination-linked glial proliferation.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating infection of the immunosuppressed brain, mediated by the gliotropic polyomavirus JCV. JCV replicates in human glial progenitor cells and astrocytes, which undergo viral T antigen-triggered mitosis, enabling viral replication. We asked if JCV spread might therefore be accelerated by glial proliferation. Using both in vitro analysis and a human glial chimeric mouse model of JCV infection, we found that dividing human astrocytes supported JCV propagation to a substantially greater degree than did mitotically quiescent cells. Accordingly, bulk and single cell RNA-sequence analysis revealed that JCV-infected glia differentially manifested cell cycle-linked disruption of both DNA damage response and transcriptional regulatory pathways. In vivo, JCV infection of humanized glial chimeras was greatly accentuated by cuprizone-induced demyelination and its associated mobilization of GPCs. Importantly, in vivo infection triggered the death of uninfected as well as infected glia, reflecting significant bystander death. Together, these data suggest that JCV propagation in PML may be accelerated by glial cell division. As such, the accentuated glial proliferation attending disease-associated demyelination may provide an especially favorable environment for JCV propagation, thus potentiating oligodendrocytic bystander death and further accelerating demyelination in susceptible hosts.

#3822 POLYOMAVIRUS INFECTION MONITORING BY QUANTITATIVE PCR IN KIDNEY RECIPIENTS AS AN INSTRUMENT FOR PREVENTING POSTTRANSPLANT COMPLICATIONS

Abstract Background and Aims Polyomaviruses (PyV) are ubiquitous human viral pathogens. BKV and JCV representing this viral family are common causative agents of viral complications among kidney recipients. Viral load higher than 1 × 107 copies/ml in urine (viruria) or 1 × 104 copies/ml in serum (viremia) in posttransplantation period may lead to polyomavirus-associated nephropathy (PVAN), hemorrhagic cystitis (HC) or even kidney transplant failure. The aim of the study was to assess PyV reactivation frequency in patients during 12 months after renal transplantation (RT) and to identify molecular subtypes of BKV and JCV. Method We examined 3207 samples of biological material (serum and urine) of 763 adult (>18 years) patients who underwent renal transplantation (RT) at the State Institution “Minsk Scientific and Practical Center for Surgery, Transplantology and Hematology”, Healthcare institutions “Brest Regional Clinical Hospital”, “Vitebsk Regional Clinical Hospital Belarus”, “Mogilev Regional Clinical Hospital”. These patients were divided into 2 groups: group 1 included 394 patients examined only for BKV infection, group 2 – 356 recipients examined for both BKV and JCV infection. Serum and urine samples for regular monitoring were collected from patients before RT, every 2 weeks first 3 months, then at 6, 9, 12 months after RT. In the case of complication development samples from patients were collected later then 1-year monitoring period. PyV DNA was detected by real-time PCR. Viral DNAs from 17 BKV-positive and 11 JCV-positive patients were molecular typed by partial sequencing of VP1 genome region. Confidence intervals for the proportions were calculated using Wald's method. Results Results showed that BKV detection total frequency in the group 1 was 14.47% [11.32%; 18.3%], almost all patients developed viruria, only 2.54% [1.32%; 4.67%] had viremia. In the group 2 PyV DNA was detected in 46.07% [40.96%; 51.26%] of recipients: 19.10% [15.34%; 23.52%] had BKV infection, 19.94% [16.11%; 24.42%] – JCV, 7.02% [4.76%; 10.2%] – BKV+JCV mixed infection. Frequency of viremia was 6.74% [4.53%; 9.87%] in this group. Maximal BKV viral load levels reached 1.2 × 1012 copies/ml in urine and 5.9 × 107 copies/ml in serum. JCV loads were up to 3 × 109 copies/ml in urine and 1.2 × 108 copies/ml in serum. Then we analyzed frequency of PyV detection before RT and during the first year after RT among the 102 recipients. Results displayed on the Fig. 1 showed that the peak of PyV infection registration and the higher risk for patient had a place on the 1.5-2.5 months after RT. Quantitative monitoring of viral load in posttransplant period was the basis for the correction of the applied immunosuppressive therapy regimens in relation to the recipients with a high viral load (higher than 1 × 107 copies/ml in urine or 1 × 104 copies/ml in serum). The results of molecular typing showed that 17 BKV isolates belonged to subgroups Ib-2 and IVc-2 (12 and 5 isolates, respectively). Within subgroups Ib-2 isolates formed 3 clusters corresponding 3 separate genovariants. JCV isolates belonged to subtype 1A, 1B and 2A (7, 3 and 1 of isolates, respectively). The last one had 99% nucleotide sequence similarity with Greece and South Korea isolates. Conclusion Our data demonstrated an importance of PyV DNA monitoring of kidney recipients in the posttransplant period starting from the first days after RT to predict development of PyV complications as PVAN, HC or others by correcting the immunosuppressive therapy.

#3822 POLYOMAVIRUS INFECTION MONITORING BY QUANTITATIVE PCR IN KIDNEY RECIPIENTS AS AN INSTRUMENT FOR PREVENTING POSTTRANSPLANT COMPLICATIONS

Abstract Background and Aims Polyomaviruses (PyV) are ubiquitous human viral pathogens. BKV and JCV representing this viral family are common causative agents of viral complications among kidney recipients. Viral load higher than 1×107 copies/ml in urine (viruria) or 1×104 copies/ml in serum (viremia) in posttransplantation period may lead to polyomavirus-associated nephropathy (PVAN), hemorrhagic cystitis (HC) or even kidney transplant failure. The aim of the study was to assess PyV reactivation frequency in patients during 12 months after renal transplantation (RT) and to identify molecular subtypes of BKV and JCV. Method We examined 3207 samples of biological material (serum and urine) of 763 adult (>18 years) patients who underwent renal transplantation (RT) at the State Institution "Minsk Scientific and Practical Center for Surgery, Transplantology and Hematology", Healthcare institutions "Brest Regional Clinical Hospital", "Vitebsk Regional Clinical Hospital Belarus", "Mogilev Regional Clinical Hospital". These patients were divided into 2 groups: group 1 included 394 patients examined only for BKV infection, group 2 – 356 recipients examined for both BKV and JCV infection. Serum and urine samples for regular monitoring were collected from patients before RT, every 2 weeks first 3 months, then at 6, 9, 12 months after RT. In the case of complication development samples from patients were collected later then 1-year monitoring period. PyV DNA was detected by real-time PCR. Viral DNAs from 17 BKV-positive and 11 JCV-positive patients were molecular typed by partial sequencing of VP1 genome region. Confidence intervals for the proportions were calculated using Wald's method. Results Results showed that BKV detection total frequency in the group 1 was 14.47% [11.32%; 18.3%], almost all patients developed viruria, only 2.54% [1.32%; 4.67%] had viremia. In the group 2 PyV DNA was detected in 46.07% [40.96%; 51.26%] of recipients: 19.10% [15.34%; 23.52%] had BKV infection, 19.94% [16.11%; 24.42%] – JCV, 7.02% [4.76%; 10.2%] – BKV+JCV mixed infection. Frequency of viremia was 6.74% [4.53%; 9.87%] in this group. Maximal BKV viral load levels reached 1.2×1012 copies/ml in urine and 5.9×107 copies/ml in serum. JCV loads were up to 3×109 copies/ml in urine and 1.2×108 copies/ml in serum. Then we analyzed frequency of PyV detection before RT and during the first year after RT among the 102 recipients. Results displayed on the fig.1 showed that the peak of PyV infection registration and the higher risk for patient had a place on the 1.5-2.5 months after RT. Quantitative monitoring of viral load in posttransplant period was the basis for the correction of the applied immunosuppressive therapy regimens in relation to the recipients with a high viral load (higher than 1×107 copies/ml in urine or 1×104 copies/ml in serum). The results of molecular typing showed that 17 BKV isolates belonged to subgroups Ib-2 and IVc-2 (12 and 5 isolates, respectively). Within subgroups Ib-2 isolates formed 3 clusters corresponding 3 separate genovariants. JCV isolates belonged to subtype 1A, 1B and 2A (7, 3 and 1 of isolates, respectively). The last one had 99% nucleotide sequence similarity with Greece and South Korea isolates. Conclusion Our data demonstrated an importance of PyV DNA monitoring of kidney recipients in the posttransplant period starting from the first days after RT to predict development of PyV complications as PVAN, HC or others by correcting the immunosuppressive therapy.

2161. Posoleucel, an Allogeneic, Off-the-Shelf Multi-Virus Specific T Cell Therapy, for Severe, Drug-Refractory Viral Infections in Pediatric Patients Following HCT: Results from a Phase 2 Trial

Abstract Background Serious viral infections are a leading cause of morbidity and mortality in pediatric recipients of allogeneic hematopoietic cell transplant (allo-HCT). Most available antivirals have significant toxicities and suboptimal efficacy. Posoleucel (PSL) is an allogeneic, off-the-shelf multi-virus specific T cell investigational product to prevent or treat 6 viral infections (AdV, BKV, CMV, EBV, HHV-6, and JCV). In the phase 2 CHARMS trial of PSL in allo-HCT recipients with intractable viral infections, 95% of patients had a clinical response. Here we present the efficacy and safety data in pediatric patients (< 18 years) enrolled in CHARMS. Methods Allo-HCT patients who had failed antiviral therapy or were unable to tolerate antivirals to control BKV, CMV, AdV, EBV, HHV-6, and/or JCV infection received a single PSL infusion of 2×107 cells/m2. Patients with a response could receive up to 4 more doses after 4 weeks, at 2-week intervals. The primary endpoint of the study was safety and feasibility. Other endpoints included complete and partial clinical response, with complete response (CR) as return to normal range and resolution of signs/symptoms and partial response (PR) defined as ≥50% decrease in viral load and/or ≥50% improvement of clinical signs/symptoms assessed by 6 weeks post infusion. Results 18 pediatric patients received PSL (1 was treated for 2 infections separately). Of the 13 with 1 viral infection, 6 (46%) had BKV, 3 (23%) CMV, 2 (15%) AdV, 2 (15%) HHV-6, and none EBV or JCV. Five (38%) had >1 infection: 3 had 2 infections, 1 had 3 infections, and 1 had 1 viral infection at each of 2 enrollments. 56% of patients received one infusion. PSL was generally well tolerated. Besides fever in 7 patients, we observed no toxicities or symptoms of CRS. Five cases of acute graft-versus-host disease (aGVHD) were observed; 3 in patients with history of aGVHD (2 had aGVHD at baseline). Both de novo cases of GVHD were Grade 1. 100% of patients achieved a clinical response (CR or PR) by 6 weeks including all 5 patients infected with multiple target viruses. Four of 6 patients with AdV infection had CR. Conclusion PSL treatment was well tolerated. All pediatric patients receiving PSL had a clinical response. Phase 3 studies including pediatric patients are in progress. Disclosures Ifigeneia Tzannou, MD, AlloVir: Advisor/Consultant Carlos Ramos, MD, Novartis: Advisor/Consultant Swati Naik, MD, Bellicum: Travel, Accomodation, Expenses Iain Fraser, MD, DPhil, AlloVir: Employee|AlloVir: Stocks/Bonds Marshelle Warren, MD, AlloVir: Advisor/Consultant Badrish Patel, MD, AlloVir: Employee|AlloVir: Stocks/Bonds Dany Ward, RN, AlloVir: Employee|AlloVir: Stocks/Bonds Ann Leen, PhD, AlloVir: Advisor/Consultant|AlloVir: Stocks/Bonds Bilal Omer, MD, AlloVir: Grant/Research Support.

JC virus-induced progressive multifocal leukoencephalopathy in a presumably healthy patient.

JC virus (JCV) is common among healthy individuals and remains latent but may be reactivated under immunosuppressive conditions, resulting in progressive multifocal leukoencephalopathy (PML). Here, we present a rare case of PML caused by JC virus infection in a previously healthy and immunocompetent patient. A 67-year-old female without any disease history was admitted after presenting with rapidly progressive dementia. The preoperative diagnosis was progressive multifocal leukoencephalopathy, and corticosteroid treatment did not improve the symptoms. Brain lesions were surgically sampled, and JCV infection was confirmed by high-throughput DNA gene detection. This patient received a combined treatment of mirtazapine, mefloquine, and traditional Chinese herbs, and had stabilization of the disease on followed-up. Although it is a rare, this case demonstrates that JC virus infection within the brain occurs in apparently healthy people. This case may increase our understanding of virus infection when facing the coronavirus epidemic in recent years, considering that similar medications were used.

Favipiravir treatment prolongs the survival in a lethal mouse model intracerebrally inoculated with Jamestown Canyon virus.

BackgroundJamestown Canyon virus (JCV) is a mosquito-borne orthobunyavirus that causes acute febrile illness, meningitis, and meningoencephalitis, primarily in North American adults. Currently, there are no available vaccines or specific treatments against JCV infections.Methodology/Principal findingsThe antiviral efficacy of favipiravir (FPV) against JCV infection was evaluated in vitro and in vivo in comparison with that of ribavirin (RBV) and 2’-fluoro-2’-deoxycytidine (2’-FdC). The in vitro inhibitory effect of these drugs on JCV replication was evaluated in Vero and Neuro-2a (N2A) cells. The efficacy of FPV in the treatment of JCV infection in vivo was evaluated in C57BL/6J mice inoculated intracerebrally with JCV, as per the survival, viral titers in the brain, and viral RNA load in the blood. The 90% inhibitory concentrations (IC90) of FPV, RBV, and 2’-FdC were 41.0, 61.8, and 13.6 μM in Vero cells and 20.7, 25.8, and 8.8 μM in N2A cells, respectively. All mice infected with 1.0×104 TCID50 died or were sacrificed within 10 days post-infection (dpi) without treatment. However, mice treated with FPV for 5 days [initiated either 2 days prior to infection (−2 dpi–2 dpi) or on the day of infection (0 dpi–4 dpi)] survived significantly longer than control mice, administered with PBS (p = 0.025 and 0.011, respectively). Moreover, at 1 and 3 dpi, the virus titers in the brain were significantly lower in FPV-treated mice (0 dpi–4 dpi) versus PBS-treated mice (p = 0.002 for both 1 and 3 dpi).Conclusions/SignificanceAlthough the intracerebral inoculation route is thought to be a challenging way to evaluate drug efficacy, FPV inhibits the in vitro replication of JCV and prolongs the survival of mice intracerebrally inoculated with JCV. These results will enable the development of a specific antiviral treatment against JCV infections and establishment of an effective animal model.

CMV, EBV, JCV and BKV infection and outcome following kidney transplantation in children initiated on a corticosteroid-minimisation immunosuppressive regimen.

Modern immunosuppressive regimens in paediatric kidney transplant recipients have contributed to improved long-term allograft survival, but at the expense of an increased incidence of viral infections. Here, we describe, for the first time, the incidence, risk factors and clinical outcome of CMV, EBV, BKV and JCV viraemia in a cohort of paediatric allograft recipients treated with a corticosteroid-minimisation immunosuppressive regimen (CMR). We retrospectively analysed 98 children treated with a CMR (basiliximab induction, corticosteroids until day 4, long-term tacrolimus and mycophenolate mofetil), who received a kidney transplant in our centre between 2009 and 2019. Over the first 4 years post-transplant, the incidences of viraemia were as follows: CMV, 25.5%; EBV, 52.0%; JCV, 16.3%; BKV, 26.5%. Younger children at time of transplant were more likely to develop EBV and BKV viraemia. EBV viraemia was also associated with a regimen involving corticosteroids, but lacking MMF. Recipient CMV serology predicted the development of EBV, BKV and CMV viraemia. Fifty-six percent of CMV viraemia episodes in high-risk patients occurred whilst the graft recipients were still receiving anti-viral prophylaxis or within 3 months of cessation. There was no difference in graft function at latest follow-up between those with and without viraemia. Judicious monitoring of viraemia, coupled with timely clinical intervention, can result in similar long-term outcomes for graft recipients compared to controls. The high incidence of CMV viraemia observed within a short period of cessation of anti-viral prophylaxis supports an extension of the length of prophylactic treatment in high-risk allograft recipients.

JC Virus Propagation is Potentiated by Glial Replication and Accelerated by Demyelination-Associated Glial Proliferation

Progressive multifocal leukoencephalopathy (PML) is a demyelinating infection of the immunosuppressed brain, mediated by the gliotropic polyomavirus JCV. JCV replicates in human glial progenitor cells and astrocytes, which undergo viral T antigen-triggered mitosis, enabling viral replication. We asked if JCV spread might therefore be accelerated by glial proliferation. We found that dividing human astrocytes supported JCV propagation more than mitotically quiescent cells. Both bulk and single cell RNA-seq revealed that these JCV-infected glia exhibited cell cycle-linked perturbation of DNA damage response and transcriptional regulatory pathways. In vivo, JCV infection of humanized glial chimeras was greatly accentuated by cuprizone-induced demyelination and its associated mobilization of GPCs. Infection triggered the death of uninfected as well as infected glia in vivo, reflecting significant bystander death. These data suggest that demyelination-associated glial proliferation accelerates the spread of JCV, and argue for the aggressive prevention of new demyelinating events in patients at risk for PML.

Treatment of Severe, Drug-Refractory Viral Infections with Allogeneic, Off-the-Shelf Multi-Virus Specific T Cells in Patients Following HSCT: Results from a Phase 2 Study

Background. Serious viral infections associated with delayed immune reconstitution are a leading cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). In approximately 90% of allogeneic HSCT patients, the suppressed immune system allows viruses that were previously in a latent, quiescent state to reactivate, with up to 60% of patients experiencing a reactivation of 2 or more viruses, including BKV, CMV, AdV, EBV and HHV-6; HSCT also predisposes for persistent acute infections, such as AdV. There is a lack of effective antiviral drugs to treat the majority of these infections and, when used clinically, available antiviral therapies are associated with significant toxicities, resulting in a substantial unmet medical need in this population. Viralym-M is an allogeneic, off-the-shelf multi-virus specific T cell (VSTs) product targeting five common viruses (BKV, CMV, AdV, EBV and HHV-6) that can lead to devastating diseases in allogeneic HSCT recipients. As T cells play a central role in response to viral infection, Viralym-M is intended to restore VST immunity to treat or prevent viral diseases and infections. Efficacy and safety of Viralym-M in allogeneic HSCT recipients with at least one treatment-refractory infection were evaluated in a phase II proof-of-concept trial (CHARMS study). In a previously published interim analysis from the study, infusion of Viralym-M was well tolerated and associated with clinical responses in most patients. Here we present the efficacy and safety data from the complete cohort of patients enrolled in the CHARMS study. Methods. Allogeneic HSCT patients who had either failed antiviral therapy or were unable to tolerate standard antivirals to control BKV, CMV, AdV, EBC, HHV-6, and/or JCV infection were enrolled to the study. Viralym-M was given as a single infusion of 2x107 VSTs/m2. If a partial response was achieved, patients could receive up to 4 additional doses after 4 weeks, at 2-week intervals. Study end points included resolution of the target infections as measured by viral load, resolution of clinical signs and symptoms as determined by the primary investigator, and safety. A complete response (CR) was defined as viral load returning to normal range and resolution of clinical signs/symptoms, while a partial response (PR) as a ≥50% decrease in viral load and/or 50% improvement of clinical signs/symptoms. Clinical end points were assessed by 6 weeks post infusion, and patients were followed until the end of study at 12 months. Persistence of the VSTs was examined by the epitope specificity of circulating T cells. Results. Fifty-eight patients were treated with Viralym-M, with one patient who was treated for two different infections separately (N=59). Nineteen (32.2%) were pediatric and 40 (67.8%) were adults. Of the patients with one viral infection, 30.5% had BKV, 28.8% CMV, 13.6% AdV, 5.1% HHV-6, 1.7% EBV, and 1.7% JCV. Eleven patients (18.6%) had more than one virus infection: 10 were co-infected with 2 different viruses, and 1 with 3 different viruses. The majority of patients received one infusion (74.6%). Overall, more than 90% of patients achieved a clinical response (PR or CR) by 6 weeks post Viralym-M infusion. 11 patients on the trial were co-infected with multiple viruses and 19 of the 23 viral infections in these patients responded to Viralym-M. We tested for persistence in 16 patients and 11 (69%) had circulating VSTs with persistence of VSTs of third party origin for up to 12 weeks. Treatment with Viralym-M was generally well tolerated. No immediate toxicities were observed and, other than isolated fever in a few patients, we observed no symptoms associated with CRS. Fourteen cases of acute graft-versus-host disease (GVHD) were observed; 8 of these were pre-existing. Of the six patients that experienced de novo GVHD, all had transient grade 1 skin GVHD that resolved with treatment. Conclusions. More than 90% of patients achieved a clinical response by 6 weeks post Viralym-M treatment, which was generally well tolerated, suggesting that Viralym-M can be safely and effectively used to treat viral infections, including multiple infections, in patients following allogeneic HSCT. Viralym-M has now received Regenerative Medicine Advanced Therapy (RMAT) and PRIority MEdicine (PRIME) designation from both the FDA and EMA. Phase 3 and proof-of-concept studies are in progress. Disclosures Tzannou: Allovir: Consultancy. Watanabe:AlloVir: Consultancy. Kuvalekar:AlloVir: Consultancy. Ramos:Novartis: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Research Funding; Kuur Therapeutics: Research Funding. Heslop:Novartis: Consultancy; Gilead Biosciences: Consultancy; PACT Pharma: Consultancy; Kiadis: Consultancy; Tessa Therapeutics: Consultancy, Research Funding; Marker Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; AlloVir: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Leen:AlloVir: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Marker Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Omer:Allovir: Consultancy, Research Funding.

The Role of the JC Virus in Central Nervous System Tumorigenesis.

Cancer is the second leading cause of mortality worldwide. The study of DNA tumor-inducing viruses and their oncoproteins as a causative agent in cancer initiation and tumor progression has greatly enhanced our understanding of cancer cell biology. The initiation of oncogenesis is a complex process. Specific gene mutations cause functional changes in the cell that ultimately result in the inability to regulate cell differentiation and proliferation effectively. The human neurotropic Polyomavirus JC (JCV) belongs to the family Polyomaviridae and it is the causative agent of progressive multifocal leukoencephalopathy (PML), which is a fatal neurodegenerative disease in an immunosuppressed state. Sero-epidemiological studies have indicated JCV infection is prevalent in the population (85%) and that initial infection usually occurs during childhood. The JC virus has small circular, double-stranded DNA that includes coding sequences for viral early and late proteins. Persistence of the virus in the brain and other tissues, as well as its potential to transform cells, has made it a subject of study for its role in brain tumor development. Earlier observation of malignant astrocytes and oligodendrocytes in PML, as well as glioblastoma formation in non-human primates inoculated with JCV, led to the hypothesis that JCV plays a role in central nervous system (CNS) tumorigenesis. Some studies have reported the presence of both JC viral DNA and its proteins in several primary brain tumor specimens. The discovery of new Polyomaviruses such as the Merkel cell Polyomavirus, which is associated with Merkel cell carcinomas in humans, ignited our interest in the role of the JC virus in CNS tumors. The current evidence known about JCV and its effects, which are sufficient to produce tumors in animal models, suggest it can be a causative factor in central nervous system tumorigenesis. However, there is no clear association between JCV presence in CNS and its ability to initiate CNS cancer and tumor formation in humans. In this review, we will discuss the correlation between JCV and tumorigenesis of CNS in animal models, and we will give an overview of the current evidence for the JC virus’s role in brain tumor formation.

Polyomaviruses BK and JC DNA infection in peripheral blood cells from blood donors

ObjectivesTo investigate the prevalence of human polyomavirus (BK and JC viruses) infection in peripheral blood mononuclear cells of healthy blood donors. MethodsThe study included 250 healthy blood donors. Five-milliliter blood was drawn into sterile EDTA tubes and PBMCs were isolated from whole blood. The isolated PBMCs were counted and stored at −70°C for future investigation. DNA was extracted and subjected to simple, sensitive and specific semi-nested PCR as well as QPCR using both general and specific primers for different assays. ResultsOf 250 blood samples, 66 (26.4%) were positive for BKV DNA (146–34,514 copies/106 cells). JC DNA was found in 45 (18%) blood samples (65–21,250 copies/106 cells). Co-infection with these viruses were found in 11 (4.4%) out of 250 blood samples. DiscussionOur study provides important data on polyomavirus infection in peripheral blood mononuclear leukocytes in immunocompetent individuals. These data indicate significant differences between the prevalence of BKV and JCV infection in healthy blood donors. The prevalence of BK and JC virus infection is higher in the age range 30–39 years compared to other age ranges.

The DNA damage response promotes polyomavirus JC infection by nucleus to cytoplasm NF- kappaB activation

BackgroundInfection of glial cells by human neurotropic polyomavirus JC (JCV), the causative agent of the CNS demyelinating disease progressive multifocal leukoencephalopathy (PML), rapidly inflicts damage to cellular DNA. This activates DNA damage response (DDR) signaling including induction of expression of DNA repair factor Rad51. We previously reported that Rad51 co-operates with the transcription factor NF-κB p65 to activate JCV early transcription. Thus Rad51 induction by JCV infection may provide positive feedback for viral activation early in JCV infection. DDR is also known to stimulate NF-κB activity, a phenomenon known as nucleus to cytoplasm or “inside-out” NF-κB signaling, which is initiated by Ataxia telangiectasia mutated (ATM) protein, a serine/threonine kinase recruited and activated by DNA double-strand breaks. Downstream of ATM, there occurs a series of post-translational modifications of NF-κB essential modulator (NEMO), the γ regulatory subunit of inhibitor of NF-κB (IκB) kinase (IKK), resulting in NF-κB activation.MethodsWe analyzed the effects of downstream pathways in the DDR by phosphospecific Western blots and analysis of the subcellular distribution of NEMO by cell fractionation and immunocytochemistry. The role of DDR in JCV infection was analyzed using a small molecule inhibitor of ATM (KU-55933). NEMO sumoylation was investigated by Western and association of ATM and NEMO by immunoprecipitation/Western blots.ResultsWe show that JCV infection caused phosphorylation and activation of ATM while KU-55933 inhibited JCV replication. JCV infection caused a redistribution of NEMO from cytoplasm to nucleus. Co-expression of JCV large T-antigen and FLAG-tagged NEMO showed the occurrence of sumoylation of NEMO, while co-expression of ATM and FLAG-NEMO demonstrated physical association between ATM and NEMO.ConclusionsWe propose a model where JCV infection induces both overexpression of Rad51 protein and activation of the nucleus to cytoplasm NF-κB signaling pathway, which then act together to enhance JCV gene expression.

JC Virus Infects Neurons and Glial Cells in the Hippocampus.

The human polyomavirus JC (JCV) infects glial cells and is the etiologic agent of the CNS demyelinating disease progressive multifocal leukoencephalopathy. JCV can infect granule cell neurons of the cerebellum, causing JCV granule cell neuronopathy and cortical pyramidal neurons in JCV encephalopathy. Whether JCV also infects neurons in other areas of the CNS is unclear. We determined the prevalence and pattern of JCV infection of the hippocampus in archival samples from 28 patients with known JCV infection of the CNS and 66 control subjects. Among 28 patients, 11 (39.3%) had JCV infection of hippocampus structures demonstrated by immunohistochemistry. Those included gray matter (dentate gyrus and cornu ammonis, subiculum) in 11/11 and afferent or efferent white matter tracts (perforant path, alveus, fimbria) in 10/11. In the hippocampus, JCV infected granule cell and pyramidal neurons, astrocytes, and oligodendrocytes. Although glial cells expressed either JCV regulatory T Antigen or JCV VP1 capsid protein, infected neurons expressed JCV T Antigen only, suggesting an abortive/restrictive infection. None of the 66 control subjects had evidence of hippocampal JCV protein expression by immunohistochemistry or JCV DNA by in situ hybridization. These results greatly expand our understanding of JCV pathogenesis in the CNS.

Early JCV infection protects against BKV infection in renal transplant recipients

Early JCV infection protects against BKV infection in renal transplant recipients

Lack of Major Histocompatibility Complex Class I Upregulation and Restrictive Infection by JC Virus Hamper Detection of Neurons by T Lymphocytes in the Central Nervous System.

The human polyomavirus JC (JCV) infects glial cells in immunosuppressed individuals, leading to progressive multifocal leukoencephalopathy. Polyomavirus JC can also infect neurons in patients with JCV granule cell neuronopathy and JCV encephalopathy. CD8-positive T cells play a crucial role in viral containment and outcome in progressive multifocal leukoencephalopathy, but whether CD8-positive T cells can also recognize JCV-infected neurons is unclear. We used immunohistochemistry to determine the prevalence of T cells in neuron-rich areas of archival brain samples from 77 patients with JCV CNS infections and 94 control subjects. Neurons predominantly sustained a restrictive infection with expression of JCV regulatory protein T antigen (T Ag), whereas glial cells were productively infected and expressed both T Ag and the capsid protein VP1. T cells were more prevalent near JCV-infected cells with intact nuclei expressing both T Ag and VP1 compared with those expressing either protein alone. CD8-positive T cells also colocalized more with JCV-infected glial cells than with JCV-infected neurons. Major histocompatibility complex class I expression was upregulated in JCV-infected areas but could only be detected in rare neurons interspersed with infected glial cells. These results suggest that isolated neurons harboring restrictive JCV infection do not upregulate major histocompatibility complex class I and thus may escape recognition by CD8-positive T cells.

Immune System Involvement in the Pathogenesis of JC Virus Induced PML: What is Learned from Studies of Patients with Underlying Diseases and Therapies as Risk Factors.

The human polyomavirus JC PyV lytic infection of oligodendrocytes in the human brain results in the demyelinating disease progressive multifocal leukoencephalopathy, PML. JCV is a common virus infection in the population that leads to PML in patients with underlying diseases and therapies that cause immune deficiencies or modulate immune system functions. Patients may have high levels of antibody to JCV that neither protect them from PML nor clear the infection once PML is established. Cell-mediated immunity plays a more effective role in clearing initial or reactivated JCV infection before PML occurs. However, patients with underlying diseases and therapies for treatment are at high risk for PML. MS patients on natalizumab are one of the categories with the highest incidence of PML. Natalizumab is a humanized monoclonal antibody targeting α4 integrins that prevents inflammatory cells from entering the brain and it has been used as a treatment for MS. A number of studies have investigated the occurrence of PML in these patients and their cell-mediated immune profile that might gain insight into the mechanism that ties natalizumab with a high risk of developing PML. It seems that cells of the immune system participate in the pathogenesis of PML as well as clearance of JCV infection.

Rad51 activates polyomavirus JC early transcription.

The human neurotropic polyomavirus JC (JCV) causes the fatal CNS demyelinating disease progressive multifocal leukoencephalopathy (PML). JCV infection is very common and after primary infection, the virus is able to persist in an asymptomatic state. Rarely, and usually only under conditions of immune impairment, JCV re-emerges to actively replicate in the astrocytes and oligodendrocytes of the brain causing PML. The regulatory events involved in the reactivation of active viral replication in PML are not well understood but previous studies have implicated the transcription factor NF-κB acting at a well-characterized site in the JCV noncoding control region (NCCR). NF-κB in turn is regulated in a number of ways including activation by cytokines such as TNF-α, interactions with other transcription factors and epigenetic events involving protein acetylation – all of which can regulate the transcriptional activity of JCV. Active JCV infection is marked by the occurrence of rapid and extensive DNA damage in the host cell and the induction of the expression of cellular proteins involved in DNA repair including Rad51, a major component of the homologous recombination-directed double-strand break DNA repair machinery. Here we show that increased Rad51 expression activates the JCV early promoter. This activation is co-operative with the stimulation caused by NF-κB p65, abrogated by mutation of the NF-κB binding site or siRNA to NFκB p65 and enhanced by the histone deacetylase inhibitor sodium butyrate. These data indicate that the induction of Rad51 resulting from infection with JCV acts through NF-κB via its binding site to stimulate JCV early transcription. We suggest that this provides a novel positive feedback mechanism to enhance viral gene expression during the early stage of JCV infection.

MicroRNA miR-J1-5p as a potential biomarker for JC virus infection in the gastrointestinal tract.

IntroductionJC virus (JCV), a human polyomavirus that causes progressive multifocal leukoencephalopathy (PML), has been linked to colorectal cancer (CRC). However, determination of JCV infection and its role in carcinogenesis has been challenging, highlighting the need for better diagnostic strategies for this virus. JCV-specific microRNAs (miRNAs) were identified and shown to negatively regulate oncogenic JCV T-Ag. Herein, we determined the pattern of JCV miRNA expression in clinical specimens from healthy subjects and CRC patients.Material and MethodsJCV miRNA expression was validated in CRC cell lines transfected with the JCV T-Ag. Results were confirmed using CRC tissues that were expressed T-Ag. Expression of JCV-specific miR-J1-5p was measured in fresh stool samples from healthy volunteers, and samples from fecal occult blood test kits from healthy subject, and patients with colorectal neoplasms.ResultsJCV miR-J1-5p was detected in JCV-transfected, but not vector-transfected, CRC cells, and was stable between cell passages. MiR-J1-5p was present in all six JCV T-Ag+ CRC samples. Surprisingly, JCV miRNA was detectable in all normal tissues, but the expression was much lower in CRC tissues. Similarly, miR-J1-5p expression was present in all fecal samples, but expression was lower in CRCs compared to controls or adenoma patients.ConclusionJC virus-specific miR-J1-5p miRNA is a potential biomarker for viral infection, and the lower expression in patients with colonic neoplasia highlights its biological role regulating oncogenic T-Ag expression in CRC.ImpactJCV-specific miRNA is a candidate for the development of a non-invasive screening test, as well as therapeutic intervention for JCV-associated diseases.

Agnogene Deletion in a Novel Pathogenic JC Virus Isolate Impairs VP1 Expression and Virion Production

Infection of glial cells by the human polyomavirus JC (JCV) causes progressive multifocal leukoencephalopathy (PML). JCV Encephalopathy (JCVE) is a newly identified disease characterized by JCV infection of cortical pyramidal neurons. The virus JCVCPN associated with JCVE contains a unique 143 base pair deletion in the agnogene. Contrary to most JCV brain isolates, JCVCPN has an archetype-like regulatory region (RR) usually found in kidney strains. This provided us with the unique opportunity to determine for the first time how each of these regions contributed to the phenotype of JCVCPN. We characterized the replication of JCVCPN compared to the prototype virus JCVMad-1 in kidney, glial and neuronal cell lines. We found that JCVCPN is capable of replicating viral DNA in all cell lines tested, but is unable to establish persistent infection seen with JCVMad-1. JCVCPN does not have an increased ability to replicate in the neuronal cell line tested. To determine whether this phenotype results from the archetype-like RR or the agnogene deletion, we generated chimeric viruses between JCVCPN of JCVMad-1. We found that the deletion in the agnogene is the predominant cause of the inability of the virus to maintain a persistent infection, with the introduction of a full length agnogene, either with or without agnoprotein expression, rescues the replication of JCVCPN. Studying this naturally occurring pathogenic variant of JCV provides a valuable tool for understanding the functions of the agnogene and RR form in JCV replication.

Gone over to the dark side

Gone over to the dark side