Noncoding Control Region Sequence Research Articles

Human papillomavirus and Merkel cell polyomavirus in Korean patients with nonsmall cell lung cancer: Evaluation and genetic variability of the noncoding control region.

Human papillomavirus (HPV) is an important causative factor of cervical cancer and is associated with nonsmall cell lung cancer (NSCLC). Merkel cell polyomavirus (MCPyV) is a rare and highly fatal cutaneous virus that can cause Merkel cell carcinoma (MCC). Although coinfection with oncogenic HPV and MCPyV may increase cancer risk, a definitive etiological link has not been established. Recently, genomic variation and genetic diversity in the MCPyV noncoding control region (NCCR) among ethnic groups has been reported. The current study aimed to provide accurate prevalence information on HPV and MCPyV infection/coinfection in NSCLC patients and to evaluate and confirm Korean MCPyV NCCR variant genotypes and sequences. DNA from 150 NSCLC tissues and 150 adjacent control tissues was assessed via polymerase chain reaction (PCR) targeting regions of the large T antigen (LT-ag), viral capsid protein 1 (VP1), and NCCR. MCPyV was detected in 22.7% (34 of 150) of NSCLC tissues and 8.0% (12 of 150) of adjacent tissues from Korean patients. The incidence rates of HPV with and without MCPyV were 26.5% (nine of 34) and 12.9% (15 of 116). The MCPyV NCCR genotype prevalence in Korean patients was 21.3% (32 of 150) for subtype I and 6% (nine of 150) for subtype IIc. Subtype I, a predominant East Asian strain containing 25 bp tandem repeats, was most common in the MCPyV NCCR data set. Our results confirm that coinfection with other tumor-associated viruses is not associated with NSCLC. Although the role of NCCR rearrangements in MCPyV infection remains unknown, future studies are warranted to determine the associations of MCPyV NCCR sequence rearrangements with specific diseases.

Detection of Merkel Cell Polyomavirus (MCPyV) DNA and Transcripts in Merkel Cell Carcinoma (MCC).

Merkel cell polyomavirus (MCPyV) is the etiological agent of the majority of Merkel cell carcinoma (MCC): a rare skin tumor. To improve our understanding of the role of MCPyV in MCCs, the detection and analysis of MCPyV DNA and transcripts were performed on primary tumors and regional lymph nodes from two MCC patients: one metastatic and one non-metastatic. MCPyV-DNA was searched by a quantitative polymerase chain reaction (qPCR), followed by the amplification of a Large T Antigen (LTAg), Viral Protein 1 (VP1) and Non-Coding Control Region (NCCR). LTAg and VP1 transcripts were investigated by reverse-transcription PCR (RT-PCR). Viral integration was also studied, and full-length LTAg sequencing was performed. qPCR revealed that the primary tumor of both patients and the lymph node of one patient was positive for the small t-antigen, with an average value of 7.0 × 102 copies/µg. The same samples harbored LTAg, NCCR and VP1 DNA. Sequencing results showed truncated LTAg with the conserved retinoblastoma (Rb) protein binding motif and VP1 and NCCR sequences identical to the MCC350 strain. RT-PCR detected LTAg but not VP1 transcripts. The MCPyV genome was integrated into the primary tumor of both patients. The results confirmed the connection between MCPyV and MCC, assuming integration, LTAg truncation and Rb sequestration as key players in MCPyV-mediated oncogenesis.

Sequence analysis of the non-coding control region of John Cunningham virus isolates from patients with multiple sclerosis treated with natalizumab

Introduction. The John Cunningham virus (JCPyV) causes a fatal demyelinating disease of the central nervous system known as progressive multifocal leukoencephalopathy (PML). In healthy people, the JCPyV non-coding control region (NCCR) is not rearranged, while NCCRs in immunocompromised patients are characterized by frequent rearrangements and can be associated with PML development. Therefore, patients treated with natalizumab, which decreases the migration of leukocytes and monocytes through the blood-brain barrier to inflammatory foci, are at increased risk of developing PML.
 The purpose of the study was to analyze NCCR sequences of JCPyV isolates from patients with multiple sclerosis (MS) treated with natalizumab.
 Materials and methods. A total of 26 blood plasma samples and 8 cerebrospinal fluid samples were analyzed using nested PCR to study the JCPyV NCCR structure in Russian MS patients treated with natalizumab. The NCCRs present in the samples were cloned and sequenced by Sanger sequencing. All the JCPyV NCCR sequences were compared with the archetype sequence and mapped. The NCCR sequences were also examined for presence of putative transcription factor binding sites.
 Results. A total of 48 NCCR sequences were found. The analysis showed that up to 55% of NCCRs were identified as rearranged NCCRs, while the other were archetype-like NCCRs. All the sequences can be divided into 6 types with one dominant rearrangement pattern. This rearranged NCCR was also found in a patient with the confirmed PML diagnosis and a poor prognosis. All the rearranged NCCRs were characterized by the presence of additional transcription factor binding sites.
 Conclusion. The study has helped identify previously unknown NCCR patterns typical of MS patients treated with natalizumab in Russia, thus confirming the need for the further research on NCCR rearrangements in MS patients undergoing natalizumab treatment to gain better understanding of the origin of neurovirulent JCPyV variants.

Rearrangement in the Hypervariable Region of JC Polyomavirus Genomes Isolated from Patient Samples and Impact on Transcription Factor-Binding Sites and Disease Outcomes.

JC polyomavirus (JCPyV) is the causative agent of the fatal, incurable, neurological disease, progressive multifocal leukoencephalopathy (PML). The virus is present in most of the adult population as a persistent, asymptotic infection in the kidneys. During immunosuppression, JCPyV reactivates and invades the central nervous system. A main predictor of disease outcome is determined by mutations within the hypervariable region of the viral genome. In patients with PML, JCPyV undergoes genetic rearrangements in the noncoding control region (NCCR). The outcome of these rearrangements influences transcription factor binding to the NCCR, orchestrating viral gene transcription. This study examines 989 NCCR sequences from patient isolates deposited in GenBank to determine the frequency of mutations based on patient isolation site and disease status. The transcription factor binding sites (TFBS) were also analyzed to understand how these rearrangements could influence viral transcription. It was determined that the number of TFBS was significantly higher in PML samples compared to non-PML samples. Additionally, TFBS that could promote JCPyV infection were more prevalent in samples isolated from the cerebrospinal fluid compared to other locations. Collectively, this research describes the extent of mutations in the NCCR that alter TFBS and how they correlate with disease outcome.

Database and Statistical Analyses of Transcription Factor Binding Sites in the Non-Coding Control Region of JC Virus

JC virus (JCV), as an archetype, establishes a lifelong latent or persistent infection in many healthy individuals. In immunocompromised patients, prototype JCV with variable mutations in the non-coding control region (NCCR) causes progressive multifocal leukoencephalopathy (PML), a severe demyelinating disease. This study was conducted to create a database of NCCR sequences annotated with transcription factor binding sites (TFBSs) and statistically analyze the mutational pattern of the JCV NCCR. JCV NCCRs were extracted from >1000 sequences registered in GenBank, and TFBSs within each NCCR were identified by computer simulation, followed by examination of their prevalence, multiplicity, and location by statistical analyses. In the NCCRs of the prototype JCV, the limited types of TFBSs, which are mainly present in regions D through F of archetype JCV, were significantly reduced. By contrast, modeling count data revealed that several TFBSs located in regions C and E tended to overlap in the prototype NCCRs. Based on data from the BioGPS database, genes encoding transcription factors that bind to these TFBSs were expressed not only in the brain but also in the peripheral sites. The database and NCCR patterns obtained in this study could be a suitable platform for analyzing JCV mutations and pathogenicity.

Merkel Cell Polyomavirus (MCPyV) in the Context of Immunosuppression: Genetic Analysis of Noncoding Control Region (NCCR) Variability among a HIV-1-Positive Population.

Background: Since limited data are available about the prevalence of Merkel cell polyomavirus (MCPyV) and the genetic variability of its noncoding control region (NCCR) in the context of immunosuppression, this study aimed to investigate the distribution of MCPyV in anatomical sites other than the skin and the behavior of NCCR among an HIV-1-positive population. Methods: Urine, plasma, and rectal swabs specimens from a cohort of 66 HIV-1-positive patients were collected and subjected to quantitative real-time polymerase chain reaction (qPCR) for MCPyV DNA detection. MCPyV-positive samples were amplified by nested PCR targeting the NCCR, and NCCRs alignment was carried out to evaluate the occurrence of mutations and to identify putative binding sites for cellular factors. Results: MCPyV DNA was detected in 10/66 urine, in 7/66 plasma, and in 23/66 rectal samples, with a median value of 5 × 102 copies/mL, 1.5 × 102 copies/mL, and 2.3 × 103 copies/mL, respectively. NCCR sequence analysis revealed a high degree of homology with the MCC350 reference strain in urine, whereas transitions, transversions, and single or double deletions were observed in plasma and rectal swabs. In these latter samples, representative GTT and GTTGA insertions were also observed. Search for putative binding sites of cellular transcription factors showed that in several strains, deletions, insertions, or single base substitutions altered the NCCR canonical configuration. Conclusions: Sequencing analysis revealed the presence of numerous mutations in the NCCR, including insertions and deletions. Whether these mutations may have an impact on the pathogenic features of the virus remains to be determined. qPCR measured on average a low viral load in the specimens analyzed, with the exception of those with the GTTGA insertion.

Exploring the role of NCCR variation on JC polyomavirus expression from dual reporter minicircles.

JC virus (JCV), a ubiquitous human polyomavirus, can cause fatal progressive multifocal leukoencephalopathy (PML) in immune compromised patients. The viral genome is composed of two conserved coding regions separated by a highly variable non-coding control region (NCCR). We analyzed the NCCR sequence from 10 PML JCV strains and found new mutations. Remarkably, the NCCR f section was mutated in most cases. We therefore explored the importance of this section in JCV expression in renal (HEK293H) and glioblastoma (U-87MG) cell lines, by adapting the emerging technology of DNA minicircles. Using bidirectional fluorescent reporters, we revealed that impaired NCCR-driven late expression in glioblastoma cells was restored by a short deletion overlapping e and f sections. This study evidenced a relevant link between JCV NCCR polymorphism and cell-type dependent expression. The use of DNA minicircles opens new insights for monitoring the impact of NCCR variation.

Novel Human Polyomavirus Noncoding Control Regions Differ in Bidirectional Gene Expression according to Host Cell, Large T-Antigen Expression, and Clinically Occurring Rearrangements.

Human polyomavirus (HPyV) DNA genomes contain three regions denoted the early viral gene region (EVGR), encoding the regulatory T-antigens and one microRNA, the late viral gene region (LVGR), encoding the structural Vp capsid proteins, and the noncoding control region (NCCR). The NCCR harbors the origin of viral genome replication and bidirectional promoter/enhancer functions governing EVGR and LVGR expression on opposite DNA strands. Despite principal similarities, HPyV NCCRs differ in length, sequence, and architecture. To functionally compare HPyV NCCRs, sequences from human isolates were inserted into a bidirectional reporter vector using dsRed2 for EVGR expression and green fluorescent protein (GFP) for LVGR expression. Transfecting HPyV NCCR reporter vectors into human embryonic kidney 293 (HEK293) cells and flow cytometry normalized to archetype BKPyV NCCR revealed a hierarchy of EVGR expression levels with MCPyV, HPyV12, and STLPyV NCCRs conferring stronger levels and HPyV6, HPyV9, and HPyV10 NCCRs weaker levels, while LVGR expression was less variable and showed comparable activity levels. Transfection of HEK293T cells expressing simian virus 40 (SV40) large T antigen (LTag) increased EVGR expression for most HPyV NCCRs, which correlated with the number of LTag-binding sites (Spearman's r, 0.625; P < 0.05) and decreased following SV40 LTag small interfering RNA (siRNA) knockdown. LTag-dependent activation was specifically confirmed for two different MCPyV NCCRs in 293MCT cells expressing the cognate MCPyV LTag. HPyV NCCR expression in different cell lines derived from skin (A375), cervix (HeLaNT), lung (A549), brain (Hs683), and colon (SW480) demonstrated that host cell properties significantly modulate the baseline HPyV NCCR activity, which partly synergized with SV40 LTag expression. Clinically occurring NCCR sequence rearrangements of HPyV7 PITT-1 and -2 and HPyV9 UF1 were found to increase EVGR expression compared to the respective HPyV archetype, but this was partly host cell type specific.IMPORTANCE HPyV NCCRs integrate essential viral functions with respect to host cell specificity, persistence, viral replication, and disease. Here, we show that HPyV NCCRs not only differ in sequence length, number, and position of LTag- and common transcription factor-binding sites but also confer differences in bidirectional viral gene expression. Importantly, EVGR reporter expression was significantly modulated by LTag expression and by host cell properties. Clinical sequence variants of HPyV7 and HPyV9 NCCRs containing deletions and insertions were associated with increased EVGR expression, similar to BKPyV and JCPyV rearrangements, emphasizing that HPyV NCCR sequences are major determinants not only of host cell tropism but also of pathogenicity. These results will help to define secondary HPyV cell tropism beyond HPyV surface receptors, to identify key viral and host factors shaping the viral life cycle, and to develop preclinical models of HPyV persistence and replication and suitable antiviral targets.

Genetic Variability of the Noncoding Control Region of Cutaneous Merkel Cell Polyomavirus: Identification of Geographically Related Genotypes.

Merkel cell polyomavirus (MCPyV) is a ubiquitous cutaneous virus that causes Merkel cell carcinoma, which develops preferentially in white populations from Europe and North America. However, the genomic variations of MCPyV among ethnic groups have not been well delineated, and even less is known regarding alterations in the noncoding control region (NCCR) in the general population. MCPyV strains recovered from skin swab specimens from 250 healthy participants with distinct ethnicities and geographic origins were subjected to sequencing analysis of the NCCR. A 25-base pair tandem repeat caused by a 25-base pair insertion within the NCCR was found predominantly in Japanese and East Asian individuals. Based on the presence of 2 other insertions and a deletion, the NCCR could be classified further into 5 genotypes. This tandem repeat was also found exclusively in the NCCR from Japanese patients with Merkel cell carcinoma, while other genotypes were detected in white patients from Europe and North America. Our results suggest that the MCPyV NCCR varies according to ethnicity and that assessing the short NCCR sequence provides a rapid and simple means for identification of the Japanese and East Asian variant genotype. It remains to be established whether these NCCR variations are associated differentially with the pathogenesis of MCPyV-driven Merkel cell carcinoma between regions with varying endemicity.

Archetype and Rearranged Non-coding Control Regions in Urothelial Bladder Carcinoma of Immunocompetent Individuals.

Polyomaviruses (PyVs) are potential transforming viruses. Despite their involvement in human tumours still being debated, there is evidence to suggest a role for PyVs in bladder carcinoma (BC). Therefore, a possible association between PyVs and BC was investigated. Urine, blood and fresh bladder tissue specimens were collected from 29 patients with BC. PyV prevalence, non-coding control region (NCCR) organization and genotypic analysis were assessed. Data showed a significant prevalence of John Cunningham (JC) PyV in BC tissues and in urine with respect to BKPyV, while simian virus 40 was not revealed. A BKPyV rearranged NCCR sequence was isolated, whereas a JCPyV archetypal structure was consistently retained. A prevalence of European genotypes was observed. Our data would suggest a JCPyV involvement in cancer progression and a BKPyV association with BC pathogenesis in immunocompetent patients. However, further work is necessary to better understand the exact role of PyVs in urothelial carcinogenesis.

Distinct BK polyomavirus non-coding control region (NCCR) variants in oral fluids of HIV- associated Salivary Gland Disease patients

HIV-associated Salivary Gland Disease (HIVSGD) is among the most common salivary gland-associated complications in HIV positive individuals and was associated with the small DNA tumorvirus BK polyomavirus (BKPyV). The BKPyV non-coding control region (NCCR) is the main determinant of viral replication and rearranges readily. This study analyzed the BKPyV NCCR architecture and viral loads of 35 immunosuppressed individuals. Throatwash samples from subjects diagnosed with HIVSGD and urine samples from transplant patients were BKPyV positive and yielded BKPyV NCCR sequences. 94.7% of the BKPyV HIVSGD NCCRs carried a rearranged OPQPQQS block arrangement, suggesting a distinct architecture among this sample set. BKPyV from HIV positive individuals without HIVSGD harbored NCCR block sequences that were distinct from OPQPQQS. Cloned HIVSGD BKPyV isolates displayed active promoters and efficient replication capability in human salivary gland cells. The unique HIVSGD NCCR architecture may represent a potentially significant oral-tropic BKPyV substrain.

Opportunistic DNA Recombination With Epstein-Barr Virus at Sites of Control Region Rearrangements Mediating JC Virus Neurovirulence

We document a unique DNA recombination between polyomavirus JC (JC virus [JCV]) and Epstein-Barr virus (EBV) at sequences of JCV found infecting the brain. Archetype JCV is present in bone marrow and uroepithelial cells of most adults. During immunosuppression, JCV can infect the brain, causing a demyelinating disease, progressive multifocal leukoencephalopathy. Rearrangements in the archetype noncoding control region are necessary for neurovirulence. Two NCCR deletions and a duplication occur at sequences of homology with EBV, present latently in B cells, which may be coinfected with both viruses. Recombination between JCV and EBV occurs in B lymphoblasts at a sequence essential for JCV neurovirulence and in cerebrospinal fluid of immunosuppressed patients with multiple sclerosis, those susceptible to progressive multifocal leukoencephalopathy. Interviral recombination is a model for conferring advantages on JCV in the brain. It can alter a critical noncoding control region sequence and potentially facilitate use of EBV DNA abilities to transfer among different cell types.

Noncoding Control Region Pattern of BK polyomavirus in Kidney Transplant Patients With Nephropathy.

Adaptation of BK polyomavirus with infected host cells may cause rearrangement of the noncoding control region of viral genomic DNA. Archetype, the prearranged transmissible form of the virus, actively replicates in the tubular epithelial cells, whereas isolates with rearranged noncoding control region sequences are found in other parts of the kidney. Clinical observations highlighted the importance of the noncoding control region rearrangements in BK virus-associated nephropathy. Therefore, we evaluated the sequence pattern of the noncoding control region in kidney transplant patients suspected of having BK virus-associated nephropathy. In this single-center, cross-sectional study, 129 kidney transplant patients suspected of having BK virus-associated nephropathy and who were admitted to Namazi Hospital were enrolled for analysis between years 2010 and 2013. Blood samples were collected from each patient. The BK polyomavirus infection was diagnosed using quantitative real-time polymerase chain reaction. The BK polyomavirus-infected patient plasma samples were amplified using in-house nested polymerase chain reaction and sequenced. The contiguous alignment noncoding control region sequences were analyzed with software. The BK polyomavirus infection was observed in plasma samples of 11 of 129 (8.5%) patients after kidney transplant. Sequence alignments showed that BK polyomavirus noncoding control region sequences in all viral infected patients with BK virus-associated nephropathy showed a complete rearranged algorithm compared with the archetype sequences. The most prevalent noncoding control region sequences were registered in a genetic sequence database (National Institutes of Health). No association was observed between risk factors and BK polyomavirus infection. There were 3 BK polyomavirus-infected patients who simultaneously had active cytomegalovirus infection. Determination of the rearranged pattern of the noncoding control region sequences in BK polyomavirus isolates from plasma samples may help improve the diagnostic and therapeutic protocols against this viral infection in patients with BK virus-associated nephropathy.

Lymphocyte Gene Expression and JC Virus Noncoding Control Region Sequences Are Linked with the Risk of Progressive Multifocal Leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML)-derived noncoding control region (NCCR) sequences permitted greater early viral gene expression than kidney-associated NCCR sequences. This was driven in part by binding of the transcription factor Spi-B to unique PML-associated Spi-B binding sites. Spi-B is upregulated in developing B cells in response to natalizumab therapy, a known risk factor for PML. Naturally occurring JCV sequence variation, together with drug treatment-induced cellular changes, may synergize to create an environment leading to an increased risk of PML.

Possible antiviral effect of ciprofloxacin treatment on polyomavirus BK replication and analysis of non-coding control region sequences

Acute renal dysfunction (ARD) is a common complication in renal transplant recipients. Multiple factors contribute to ARD development, including acute rejection and microbial infections. Many viral infections after kidney transplantation result from reactivation of “latent” viruses in the host or from the graft, such as the human Polyomavirus BK (BKV). We report the case of a 39 year-old recipient of a 2nd kidney graft who experienced BKV reactivation after a second episode of acute humoral rejection. A 10-day treatment with the quinolone antibiotic ciprofloxacin was administered with an increase of immunosuppressive therapy despite the active BKV replication. Real Time PCR analysis performed after treatment with ciprofloxacin, unexpectedly showed clearance of BK viremia and regression of BK viruria. During the follow-up, BK viremia persisted undetectable while viruria decreased further and disappeared after 3 months.BKV non-coding control region sequence analysis from all positive samples always showed the presence of archetypal sequences, with two single-nucleotide substitutions and one nucleotide deletion that, interestingly, were all representative of the subtype/subgroup I/b-1 we identified by the viral protein 1 sequencing analysis.We report the potential effect of the quinolone antibiotic ciprofloxacin in the decrease of the BKV load in both blood and urine.

Quasispecies Analysis of JC Virus DNA Present in Urine of Healthy Subjects

JC virus is a human polyomavirus that infects the majority of people without apparent symptoms in healthy subjects and it is the causative agent of progressive multifocal leucoencephalopathy (PML), a disorder following lytic infection of oligodendrocytes that mainly manifests itself under immunosuppressive conditions. A hallmark for JC virus isolated from PML-brain is the presence of rearrangements in the non-coding control region (NCCR) interspersed between the early and late genes on the viral genome. Such rearrangements are believed to originate from the archetype JC virus which is shed in urine by healthy subjects and PML patients. We applied next generation sequencing to explore the non-coding control region variability in urine of healthy subjects in search for JC virus quasispecies and rearrangements reminiscent of PML. For 61 viral shedders (out of a total of 254 healthy subjects) non-coding control region DNA and VP1 (major capsid protein) coding sequences were initially obtained by Sanger sequencing. Deletions between 1 and 28 nucleotides long appeared in ∼24.5% of the NCCR sequences while insertions were only detected in ∼3.3% of the samples. 454 pyrosequencing was applied on a subset of 54 urine samples demonstrating the existence of JC virus quasispecies in four subjects (∼7.4%). Hence, our results indicate that JC virus DNA in urine is not always restricted to one unique virus variant, but can be a mixture of naturally occurring variants (quasispecies) reflecting the susceptibility of the non-coding control region for genomic rearrangements in healthy individuals. Our findings pave the way to explore the presence of viral quasispecies and the altered viral tropism that might go along with it as a potential risk factor for opportunistic secondary infections such as PML.

Polyomavirus JC in the Context of Immunosuppression: A Series of Adaptive, DNA Replication-Driven Recombination Events in the Development of Progressive Multifocal Leukoencephalopathy

Polyomavirus JC (JCV) is the etiological agent of progressive multifocal leukoencephalopathy (PML), a demyelinating infection of oligodendrocytes in the brain. PML, a frequently fatal opportunistic infection in AIDS, has also emerged as a consequence of treatment with several new immunosuppressive therapeutic agents. Although nearly 80% of adults are seropositive, JCV attains an ability to infect glial cells in only a minority of people. Data suggest that JCV undergoes sequence alterations that accompany this ability, and these changes can be derived from an archetype strain by mutation, deletion, and duplication. While the introductory source and primary tissue reservoir of JCV remain unknown, lymphoid cells have been identified as potential intermediaries in progression of JCV to the brain. This review is focused on sequence changes in the noncoding control region (NCCR) of the virus. We propose an adaptive mechanism that involves a sequential series of DNA replication-driven NCCR recombination events involving stalled DNA replication forks at NCCR palindromic secondary structures. We shall describe how the NCCR sequence changes point to a model in which viral DNA replication drives NCCR recombination, allowing JCV adaptation to different cell types in its progression to neurovirulence.

Sequential changes in the non-coding control region sequences of JC polyomaviruses from the cerebrospinal fluid of patients with progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is caused by JC polyomavirus (JCV) infection in the brain. JCV isolates from PML patients have variable mutations in the non-coding control region (NCCR) of the genome. This study was conducted to examine sequential changes in NCCR patterns of JCV isolates obtained from the cerebrospinal fluid (CSF) of PML patients. CSF specimens were collected from PML patients at different time points, the NCCR sequences were determined, and their compositions were assessed by computer-based analysis. In patients showing a marked increase in JCV load, the most frequent NCCR sequences in the follow-up specimens were different from those in the initial samples. In contrast, the dominant NCCRs in the CSF remained unaltered during the follow-up of individuals in whom the viral load decreased after therapeutic intervention. These data demonstrate that the majority of JCV variants emerge with the progression of PML and that these changes are suppressed when the viral load is decreased.

Reactivation of human polyomavirus JC in patients affected by psoriasis vulgaris and psoriatic arthritis and treated with biological drugs: Preliminary results

Psoriasis vulgaris (PsV) and psoriatic arthritis (PSA) are inter-related heritable inflammatory skin diseases. Psoriatic lesions develop as a result of abnormal immune responses, hyperproliferation and altered differentiation of keratinocytes, and a notable subset of psoriatic patients develops PsA, characterized by joints inflammation. Recently, biological drugs were introduced to treat these diseases. However, this therapy has already been associated with the development of serious life-threatening infections, such as the reactivation of human polyomavirus JC (JCV), responsible for the progressive multifocal leukoencephalopathy (PML), a lethal demyelinating disease caused by oligodendrocytes lytic infection. Therefore, the aims of our study were the investigation of the possible JCV reactivation in PsV and PsA patients treated with adalimumab, etanercept, and methotrexate, performing quantitative real-time PCR in sera and skin biopsies at the time of recruitment (T0) and after 3 (T3) and 6 (T6) months of treatment, and the sequencing analysis of the JCV non-coding control region (NCCR). We found JCV DNA in 5/15 PsV patients and in 2/15 PsA patients and JCV NCCR sequence analysis always showed a structure similar to non-pathogenic CY archetype, with random occurrence of a few irrelevant point mutations. Nevertheless the poor number of patients analyzed, our preliminary data can pave the way for taking into account that the follow-up of JCV DNA detection and the JCV NCCR sequence analysis in psoriatic patients may be important to evaluate the risk of PML onset, considering that patients affected by autoimmune diseases and treated with biologics continue to rise.

Phylogenetic analysis of the complete genome of 11 BKV isolates obtained from allogenic stem cell transplant recipients in Ireland

BK polyomavirus (family Polyomaviridae) may cause hemorrhagic cystitis (BKV-HC) in hematopoietic stem cell transplant recipients. Eleven complete BKV genomes (GenBank accession numbers: JN192431-JN192441) were sequenced from urine samples of allogenic hematopoietic stem cell transplant recipients and compared to complete BKV genomes in the published literature. Of the 11 isolates, seven (64%) were subgroup Ib-1, three (27%) isolates belonged to subgroup Ib-2 and a single isolate belonged to subtype III. The analysis of single-nucleotide polymorphisms in this study showed that isolates could be subclassified into subtypes I-IV and subgroups Ib-1 and Ib-2 on the basis of VP1 of the first part of the Large T-antigen (LTag). The non-coding control region (NCCR) of the 11 isolates was also sequenced. These sequences showed that there was consistent sequence homology within subgroups Ib-1 and Ib-2. Two new mutations were described in the isolates, G→C at O(84) in isolate SJH-LG-310, and a deletion at R(2-7) in isolate SJH-LG-309. No known transcription factor is thought to be present at the site of either of these mutations. There were no rearrangements seen in isolates and this may be because the patients were not followed up over time. There were five nucleotide positions at which subgroup Ib-1 isolated differed from subgroup Ib-2 isolates in the NCCR sequence, O(41) , P(18) , P(31) , R(4) , and S(18) . The mutation O(41) is present in the promoter granulocyte/macrophage stimulating factor) gene and the P(31) mutation is present in the NF-1 gene.