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Polyomavirus BK (BKPyV) T-antigens (large and small tumor antigens, or Lt-ag and st-ag, respectively), control key aspects of viral replication and are able to regulate cell cycle, promoting cell proliferation. However, the structural effects of genetic mutations on T-antigens are poorly investigated. In this study, 214 sequences of T-antigens from individuals with different BKPyV infections (16 renal transplant with nephropathy; 78 asymptomatic renal transplant; 24 hematopoietic stem cell transplant with hemorrhagic cystitis; 96 healthy non-transplant), were analyzed from the genetic and structural standpoints. We found a high concentration of non-synonymous mutations at inter-domains and hexamerization regions of both proteins, being five of them under positive selection in the Lt-ag but none in the st-ag. The in silico analysis indicated that two mutations, located at positions 164 in the st-ag and 592 in the Lt-ag, would significantly affect the interaction with PP2A and p53 cell targets, respectively, although they were not associated to a specific clinical status. No mutations were detected on the J-domains or at the ATPase motif. In sum, the profile of the mutations found seem not to be associated to increased morbidity. This is the first work to analyze structural modifications on T-antigens in different BKPyV infections, and managed to map conserved and variable regions of the T-antigens, which will be helpful for the study of new antiviral drugs. J. Med. Virol. 87:1418–1426, 2015. © 2015 Wiley Periodicals, Inc.

Very little is known about JCV infection and genotype distribution with respect to the different demographic and clinical characteristics of the Italian population. A cross-sectional study was carried out on the prevalence of JCV genotypes in 323 Caucasian subjects (mean age: 37.5 years, range: 2-70 years). Urine samples from 200 immunocompromised patients, including patients affected by Multiple Sclerosis (MS), Human Immunodeficiency Virus (HIV), colon cancer, inflammatory diseases and Progressive Multifocal Leukoencephalopathy (PML), and 123 immunocompetent individuals were tested by quantitative real time PCR. Sequencing of the JCV viral protein 1 (VP1) and transcriptional control region (TCR) was performed. In this series, the overall prevalence of JCV excretion was 32.8% without significant differences between males and females. JCV was detected in 39.5% of patients and in 22% of healthy individuals (P = 0.004). The most prevalent JCV genotype excreted was genotype 1 (69.8%), followed by genotype 2 (22.6%) and genotype 4 (7.5%). Distribution of genotypes between patients and healthy subjects showed a statistically significant difference for the type 1 compared to the other variants (P

JC virus (JCV) is a polyomavirus that asymptomatically infects up to 80% of the worldwide human population and establishes latency in the kidney. In the case of host immunodeficiency, it can cause progressive multifocal leukoencephalopathy (PML), which is a fatal demyelinating disease of the central nervous system. In an attempt to understand better PML pathogenesis and JCV infection, the presence of the JCV genome and expression of the early viral protein in the brain of deceased individuals, with and without HIV infection, was investigated. Sixty autopsy samples of brain tissues were collected from 15 HIV-positive PML patients, 15 HIV-positive patients with other neurological diseases, 15 HIV-positive patients without neurological disorders, and 15 HIV-negative individuals who died from diseases unrelated to the central nervous system. By means of specific Real Time Polymerase Chain Reaction, the JCV genome was detected in 14 of 15 PML brains, three of 15 HIV-positive brains (with and without neurological diseases), and 1 of 15 HIV-negative brains. JCV genotyping was also performed. Expression of the early JCV protein T Antigen was verified by a specific immunohistochemistry assay, and it was found in the brain tissues from 12 PML cases and one case with other neurological disease. The data obtained demonstrate that infection of the brain with JCV can also be observed in the brains of HIV-negative individuals, without neurological disorders. However, viral protein expression was limited to PML brains and to one brain from a patient with other neurological disease, suggesting that JCV can also be present in the brains of patients without PML.

The link between human herpesvirus 8 (KSHV) and Kaposi's sarcoma (KS) has been proven, but many important aspects including risk factors, genetic predisposition to tumor development, transmission of KSHV, and the pathogenic potential of different genotypes remain to be elucidated. Possible associations between clinical parameters and antibody levels, viral load fluctuations, and viral genotype were analyzed by quantitative real-time PCR, an in-house developed IFA assay, and sequence analysis of ORF K1-VR1 in blood, serum and saliva of 38 subjects with classic KS (cKS). KSHV lytic antibodies were significantly increased in stage IV compared to stage I and II patients (p = 0.006 and p = 0.041, respectively). KSHV blood, serum, and saliva viral load was comparable in all stages. The highest viral loads were detected in saliva, and they decreased in stages III-IV compared to stages I-II patients. Higher concentrations of lytic antibodies and higher viral loads were observed in fast progressing cKS patients, in whom KSHV detection from blood was also more frequent. Type A KSHV strain was almost exclusively present in rapid progressors (12/17 cases), while C type was mainly present in slow progressing patients (6/7 cases). Finally, detection of type A KSHV strain associated with higher blood viral loads. KSHV lytic antibody levels and viral load can be used to monitor clinical evolution of cKS. Infection supported by KSHV A subtype is associated with more rapid progressive disease. Careful monitoring and aggressive therapeutic protocols should be considered in patients with KSHV A-supported infection.

Polyomavirus BK (BKPyV) T-antigens (large and small tumor antigens, or Lt-ag and st-ag, respectively), control key aspects of viral replication and are able to regulate cell cycle, promoting cell proliferation. However, the structural effects of genetic mutations on T-antigens are poorly investigated. In this study, 214 sequences of T-antigens from individuals with different BKPyV infections (16 renal transplant with nephropathy; 78 asymptomatic renal transplant; 24 hematopoietic stem cell transplant with hemorrhagic cystitis; 96 healthy non-transplant), were analyzed from the genetic and structural standpoints. We found a high concentration of non-synonymous mutations at inter-domains and hexamerization regions of both proteins, being five of them under positive selection in the Lt-ag but none in the st-ag. The in silico analysis indicated that two mutations, located at positions 164 in the st-ag and 592 in the Lt-ag, would significantly affect the interaction with PP2A and p53 cell targets, respectively, although they were not associated to a specific clinical status. No mutations were detected on the J-domains or at the ATPase motif. In sum, the profile of the mutations found seem not to be associated to increased morbidity. This is the first work to analyze structural modifications on T-antigens in different BKPyV infections, and managed to map conserved and variable regions of the T-antigens, which will be helpful for the study of new antiviral drugs.

In order to investigate the possible involvement of viruses in Multiple Sclerosis (MS), the study evaluated the presence of viral genomic sequences in cerebrospinal fluid (CSF), as markers of viral replication within the central nervous system (CNS). A total of 85 CSF samples were collected from 38 MS patients, 28 patients with other neurological diseases and 19 subjects without neurological diseases. Using nested-PCR, the investigation focused on the presence of human herpes virus DNA, including herpes simplex virus 1 (HSV-1) and 2 (HSV-2), the Epstein-Barr virus (EBV), varicella zoster virus (VZV), human cytomegalovirus (HCMV), human herpes virus 6 (HHV-6) and JC virus (JCV). All the CSF samples from the individuals without neurological diseases were negative for viral DNA. Genomic sequences of HSV-1, HCMV, EBV, HHV6, and JCV were found in patients with MS and other neurological diseases without significant differences between the two groups. VZV DNA was detected more frequently (P < 0.05) in the MS group (31.6%), particularly among the relapsing-remitting MS patients (43.5%), compared with patients with other neurological diseases (10.7%). In addition, the results indicated that JCV and HHV-6 were replicating actively in the CNS of a small, but significant number of patients with MS and other neurological diseases. Most importantly, the study revealed a high frequency of VZV DNA in the CSF of patients with MS, suggesting a possible role of this virus in the pathogenesis of MS.

The etiology of brain tumors and meningiomas is still unknown. Several factors have been considered, such as genetic predisposition and environmental risk factors, but the hypothesis that one or more infectious agents may play a role in tumor pathogenesis has also been investigated. Therefore, emphasis was placed on the neurooncogenic family Polyomaviridae and the presence of human polyomavirus DNA sequences and JCV mRNA were examined in malignant human brain biopsies. Italian patients affected with different types of neoplasias of the brain and its covering were enrolled. The patients underwent surgical tumor excision and the presence of the polyomavirus genome in biopsy and other body fluids was evaluated by PCR. In addition, the genomic organization of JCV was examined in depth, with the aim of providing information on genotype distribution and TCR rearrangements in the population affected with intracranial neoplasms. On the whole, polyomavirus DNA was found in 50% of the biopsy specimens studied, JC virus DNA and BK virus DNA were amplified in 40.6% mainly glioblastomas and 9.4% of the tissue specimens, respectively, while none of the biopsy specimens tested contained Simian virus 40 DNA. Genotype 1 and Mad 4 TCR organization were the most frequent in the population enrolled. Although a cause and effect was not demonstrated and the specific role of the viruses remains unknown, the findings appear to confirm the hypothesis that JCV and BKV could be important co-factors in tumor pathogenesis. J. Med. Virol. 77:447–454, 2005. © 2005 Wiley-Liss, Inc.

This is a case study of a child who developed roseola infantum first, then varicella, and was later affected by acute cerebellar syndrome, severe truncal ataxia, and myoclonic dystonia. Human herpesvirus 6 (HHV-6) A and B were detected in the cerebrospinal fluid (CSF) and peripheral blood, respectively, upon ataxia onset. The intricacy of this case suggests multifaceted conclusions ranging from the need for a multidirectional approach to neurological diseases, to confirmation of a more pronounced neurotropism of HHV-6A and a possible role of viruses in myoclonic dystonia syndrome, although this last hypothesis should be confirmed by larger studies.

Very little is known about JCV infection and genotype distribution with respect to the different demographic and clinical characteristics of the Italian population. A cross-sectional study was carried out on the prevalence of JCV genotypes in 323 Caucasian subjects (mean age: 37.5 years, range: 2-70 years). Urine samples from 200 immunocompromised patients, including patients affected by Multiple Sclerosis (MS), Human Immunodeficiency Virus (HIV), colon cancer, inflammatory diseases and Progressive Multifocal Leukoencephalopathy (PML), and 123 immunocompetent individuals were tested by quantitative real time PCR. Sequencing of the JCV viral protein 1 (VP1) and transcriptional control region (TCR) was performed. In this series, the overall prevalence of JCV excretion was 32.8% without significant differences between males and females. JCV was detected in 39.5% of patients and in 22% of healthy individuals (P = 0.004). The most prevalent JCV genotype excreted was genotype 1 (69.8%), followed by genotype 2 (22.6%) and genotype 4 (7.5%). Distribution of genotypes between patients and healthy subjects showed a statistically significant difference for the type 1 compared to the other variants (P 0.01). Of note, JCV genotype 2 was found to be associated to young patients (P = 0.0001) and to patients treated with immunomodulator drugs (P = 0.0001), but not to PML subjects. The non-pathogenic archetype IIS (singular, insert) form was present in all JCV strains detected. This result allows to hypothesize a possible JCV genotype selection in response to pressure by immunomodulatory drugs.

Treatment with biological drugs is associated with increased susceptibility to viral infections. Reactivation of JC virus (JCV) and human cytomegalovirus (HCMV) in adults after therapy has been documented. The long-term effects of biological and conventional therapy on human herpesviruses and polyomaviruses infections in young patients were assessed. One hundred eighty-six samples [urine, serum, and blood cells (PBMCs)] from 62 patients (15.8 ± 6.2 years old) with Crohn's disease, ulcerative rectocolitis or juvenile rheumatoid arthritis treated with immunotherapy or conventional therapy for over 12 months were tested by real time PCR. One hundred twenty-four samples (urine and blood) from 62 matched healthy volunteers (13.8 ± 8.6 years old) were included as controls. Sequencing of the JCV viral protein 1 (VP1) and transcriptional control region (TCR) was performed. Herpes simplex virus 1/2 and varicella zoster virus genomes were not detected in any patients, whereas Epstein-Barr virus, HCMV, and human herpesvirus-6 genomes were detected in 4.8%, 3.2%, and 1.6% of the patients, respectively. JCV was detected in 22.6% (14/62) of urine samples from patients and in 8% (5/62) from controls, in 50% (7/14) of sera from patients shedding JCV, and in 71.4% (5/7) of matched PBMCs. There was a significant association between infliximab treatment and excretion of JCV genotype 2. Subclinical infection/reactivation of JCV genotype 2 in young patients during infliximab therapy was demonstrated. Conversely, increased susceptibility to herpesviruses infection was not shown. Future studies are warranted to investigate the effects of JCV reactivation on the health of young patients treated with infliximab.

Rituximab is a chimeric monoclonal antibody reacting with the CD20 antigen on B cells. It has been proposed as treatment for the idiopathic nephrotic syndrome, recurrent idiopathic nephropathy, and focal segmental glomerulosclerosis refractory to steroids. Rituximab influences T-cell immunity and may predispose the patients to opportunistic infections, such as progressive multifocal leukoencephalopathy caused by the polyomavirus JC (JCV). The risk of latent viruses infections/reactivations in pediatric patients receiving monoclonal antibodies is not well known yet. In this longitudinal 6-month study, the effects of rituximab on JCV and BK virus (BKV) replication have been investigated. Blood, serum, and urine samples have been collected monthly from 11 pediatric patients (mean age: 11 years) with the idiopathic nephrotic syndrome and recurrent idiopathic nephropathy, under rituximab therapy. JCV and BKV real-time PCRs and sequencing of the viral protein 1 and the non-coding control region have been conducted. The same investigations have been undertaken on samples collected from eight pediatric patients (controls, mean age: 6 years), with idiopathic nephrotic syndrome or focal segmental glomerulosclerosis, treated with conventional chemotherapy. JCV was detected in the urine of one patient (9%), and one control (12.5%); BKV was found in the urine of 7/11 patients (63.6%) and 2/8 controls (25%) and in blood samples from four patients. No significant difference was found in the mean viral loads and in the viral molecular characterizations between the two groups. The polyomaviruses replication was not associated with rituximab therapy in children. J. Med. Virol. 84:1464–1470, 2012. © 2012 Wiley Periodicals, Inc.

The role of viruses in the pathogenesis of multiple sclerosis (MS) is a subject of heated debate. The presence of six different neurotropic viruses was sought, including JC virus (JCV), varicella zoster virus (VZV), human herpesvirus 6 (HHV-6), and Epstein-Barr virus (EBV), in cerebrospinal fluid (CSF) samples collected from 51 patients with MS and 30 patients with other neurological diseases. Cell-free or cell-associated viral DNA in CSF samples was detected by real-time PCR, and viral loads were determined. Magnetic resonance imaging (MRI) examinations were also performed to look for active lesions. Cell-associated JCV DNA was detected in 3 of the 51 patients with MS and in 2 of the 30 patients with other neurological disease. Cell-free JCV DNA was detected in one additional patient with MS. Cell-free VZV DNA was detected in one patient without MS, cell-free HHV-6 was detected in one patient with MS, and cell-free EBV was detected in one patient with MS. All other study patients had no detectable viral DNA in CSF samples and no double infections were found. The small percentage of patients with detectable viral DNA in CSF samples was comparable between patients with MS and those with other neurological disease, and presence of viral DNA was not a predictor of brain lesions. Additional observations suggest that cell trafficking from the periphery, rather than leakage through the blood-brain barrier, results in the transport of viruses to the CNS, where local immunosurveillance can control viral replication in immunocompetent individuals.

Human polyomavirus JC (JCV) infects the worldwide population, remains latent in the kidney, and is excreted in the urine. A longitudinal study was performed in order to evaluate JCV excretion, to characterize molecularly the virus and to determine if its presence in urine is a consequence of viral reactivation or merely of epithelial squamous cell shedding. The presence of cellular sediment and the JCV genome were examined in 333 urine samples collected periodically for 3 months from 17 healthy subjects; molecular characterization, and quantitation of the virus were also undertaken. JCV DNA was detected in 40.2% of the samples, with a significant difference (P