Publications by authors named "Eeva Auvinen"

41 Publications

Distribution of HPV Genotypes Differs Depending on Behavioural Factors among Young Women.

Microorganisms 2021 Apr 2;9(4). Epub 2021 Apr 2.

Department of Obstetrics and Gynecology, Tampere University Hospital and Tampere University, 33100 Tampere, Finland.

Risk factors for the different human papillomavirus (HPV) genotypes are not well understood, although the risk of cancer is known to vary among them. Our aim was to evaluate the association of diverse behavioral and reproductive factors with genotype-specific HPV prevalence among 879 unvaccinated women aged 18-75 years referred to the colposcopy clinic at Helsinki University Hospital in Finland. Cervical swabs for HPV genotyping were collected in the first visit and assessed for 34 high-risk (hr) and low-risk (lr) HPV genotypes. Participants completed a questionnaire on behavioral, reproductive, and lifestyle factors. Differences in genotype-specific HPV prevalence were analyzed overall and in age groups using binary logistic regression. Smoking was associated with higher prevalence in HPV16 compared with other hrHPV genotypes together with decreasing age, being highest among younger women <30 years old, odds ratio (OR) 3.74 (95% CI 1.42-9.88). The later the sexual debut, the more it seemed to protect from HPV16 infection. The best protection was achieved when the sexual debut took place at >20 years of age, with an OR of 0.43 (95% CI 0.23-0.83). This association was not seen with other hrHPV genotypes. Methods of contraception seemed not to have an effect on hrHPV positivity, regardless of the HPV genotype. The genotype specific hrHPV prevalence differs, depending on behavioral factors, especially among younger women referred to colposcopy.
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http://dx.doi.org/10.3390/microorganisms9040750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8066411PMC
April 2021

Recommendations for the introduction of metagenomic high-throughput sequencing in clinical virology, part I: Wet lab procedure.

J Clin Virol 2021 01 18;134:104691. Epub 2020 Nov 18.

Department of Microbiology and Ecology, Faculty of Medicine, University of Valencia, Valencia, Spain. Electronic address:

Metagenomic high-throughput sequencing (mHTS) is a hypothesis-free, universal pathogen detection technique for determination of the DNA/RNA sequences in a variety of sample types and infectious syndromes. mHTS is still in its early stages of translating into clinical application. To support the development, implementation and standardization of mHTS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mHTS for viral diagnostics to share methodologies and experiences, and to develop application recommendations. This manuscript aims to provide practical recommendations for the wet lab procedures necessary for implementation of mHTS for virus diagnostics and to give recommendations for development and validation of laboratory methods, including mHTS quality assurance, control and quality assessment protocols.
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http://dx.doi.org/10.1016/j.jcv.2020.104691DOI Listing
January 2021

Progressive Multifocal Leukoencephalopathy: Current Insights.

Degener Neurol Neuromuscul Dis 2019 2;9:109-121. Epub 2019 Dec 2.

Clinical Neurosciences, Neurology, Helsinki University Hospital and Helsinki University, Helsinki, Finland.

Cases of PML should be evaluated according to predisposing factors, as these subgroups differ by incidence rate, clinical course, and prognosis. The three most significant groups at risk of PML are patients with hematological malignancies mostly previously treated with immunotherapies but also untreated, patients with HIV infection, and patients using monoclonal antibody (mAb) treatments. Epidemiological data is scarce and partly conflicting, but the distribution of the subgroups appears to have changed. While there is no specific anti-JCPyV treatment, restoration of the immune function is the most effective approach to PML treatment. Research is warranted to determine whether immune checkpoint inhibitors could benefit certain PML subgroups. There are no systematic national or international records of PML diagnoses or a risk stratification algorithm, except for MS patients receiving natalizumab (NTZ). These are needed to improve PML risk assessment and to tailor better prevention strategies.
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http://dx.doi.org/10.2147/DNND.S203405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6896915PMC
December 2019

Next-generation sequencing shows marked rearrangements of BK polyomavirus that favor but are not required for polyomavirus-associated nephropathy.

J Clin Virol 2020 01 13;122:104215. Epub 2019 Nov 13.

Department of Virology and Immunology, Helsinki University Hospital Laboratory, Helsinki, Finland; Department of Virology, University of Helsinki, Helsinki, Finland. Electronic address:

Background: BKPyV is associated with polyomavirus-associated nephropathy (PVAN), a major cause of graft rejection in kidney transplant recipients (KTRs). Mutations occur in the transcriptional control region (TCR) of BKPyV, but whether they are required for the development of PVAN is not completely understood. To this end, we characterized BKPyV TCRs from KTRs to assess whether TCR mutations are associated with PVAN.

Study Design: We analyzed urine and plasma samples of fifteen KTRs with biopsy-confirmed PVAN, presumptive PVAN, or probable PVAN in order to explore the contents of the BKPyV virome. BKPyV TCRs were amplified and deep sequenced to characterize the viral strains. Alterations in block structures and transcription factor binding sites were investigated.

Results: The majority of sequences in both urine and plasma samples represented archetype BKPyV TCR. Minor populations harboring rearranged TCRs were detected in all patient groups. In one biopsy-confirmed PVAN patient rearranged TCRs predominated, and in another patient half of all reads represented rearranged sequences.

Conclusions: Although archetype BKPyV predominated in most patients, highest proportions and highest numbers of rearranged strains were detected in association with PVAN. TCR mutations seem not necessary for the development of PVAN, but immunosuppression may allow increased viral replication giving rise to TCR variants with enhanced replication efficiency.
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http://dx.doi.org/10.1016/j.jcv.2019.104215DOI Listing
January 2020

BetaHPV E6 and E7 colocalize with NuMa in dividing keratinocytes.

Virus Genes 2019 Oct 9;55(5):600-609. Epub 2019 Jul 9.

University of Helsinki and Helsinki University Hospital Laboratory, Helsinki, Finland.

Human papillomaviruses (HPVs) of genus betapapillomavirus (betaHPV) are implicated in skin carcinogenesis, but their exact role in keratinocyte transformation is poorly understood. We show an interaction of HPV5 and HPV8 oncoproteins E6 and E7 with the nuclear mitotic apparatus protein 1 (NuMA). Binding of E6 or E7 to NuMA induces little aneuploidy, cell cycle alterations, or aberrant centrosomes. Intracellular localization of NuMA is not altered by E6 and E7 expression in 2D cultures. However, the localization profile is predominantly cytoplasmic in 3D organotypic skin models. Both viral proteins colocalize with NuMA in interphase cells, while only E7 colocalizes with NuMA in mitotic cells. Intriguingly, a small subset of cells shows E7 at only one spindle pole, whereas NuMA is present at both poles. This dissimilar distribution of E7 at the spindle poles may alter cell differentiation, which may in turn be relevant for betaHPV-induced skin carcinogenesis.
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http://dx.doi.org/10.1007/s11262-019-01685-9DOI Listing
October 2019

BK polyomavirus viremia and antibody responses of pediatric kidney transplant recipients in Finland.

Pediatr Transplant 2019 02 16;23(1):e13324. Epub 2018 Nov 16.

Children's Hospital, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.

Background: BKPyV is an important cause of premature graft failure after KT. Most clinical studies describe BKPyV infection in adult KT patients. We studied the prevalence of post-transplant BKPyV viremia, serology, and graft function in pediatric KT recipients.

Methods: Forty-six pediatric patients transplanted between 2009 and 2014 were followed up for BKPyV DNAemia by plasma PCR for median 2.3 (range: 1-6) years. BKPyV-specific antibodies were retrospectively analyzed using virus-like particle ELISA. GFR was measured annually by Cr-EDTA clearance, and serum samples were screened for DSAs by Luminex assay.

Results: BKPyV viremia was demonstrated in nine patients at a median of 6 months post-KT. Early BKPyV viremia at 3 months post-KT associated with decreased concomitant GFR and tendency for decreased subsequent graft function. Three of nine patients with BKPyV viremia developed DSA, all against class II antigens. PyVAN developed to four patients and responded to judicious reduction in IS. One graft was lost later due to ABMR. BKPyV-IgG was found in 18 of 31 patients (58%) tested at transplantation, and seven recipients seroconverted after transplantation with a significant increase in IgG levels with IgM. Finally, BKPyV-IgG was detectable in 31 of 40 patients (78%) at the end of the study.

Conclusions: Post-transplant BKPyV viremia in pediatric KT patients may alter graft function and contribute to progression of chronic allograft injury. BKPyV-IgG predicts past exposure. Low or absent BKPyV-specific antibody levels were seen pretransplant in 42% of tested patients, but were not predictive of prolonged replication or poor outcome.
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http://dx.doi.org/10.1111/petr.13324DOI Listing
February 2019

Genotyping of hepatitis C virus by nucleotide sequencing: A robust method for a diagnostic laboratory.

MethodsX 2018 16;5:414-418. Epub 2018 Apr 16.

Department of Virology, University of Helsinki and Helsinki University Hospital Laboratory, Helsinki, Finland.

Hepatitis C virus (HCV) is a globally significant blood-borne agent causing liver diseases, and it has infected over 170 million people worldwide. HCV is a diverse group of RNA viruses currently divided into genotypes 1-7 as well as subtypes. HCV infection can be treated with antiviral drugs, but the HCV genotype has to be determined for optimal selection of treatment strategy. The aim of this study was to set up a sequencing-based HCV genotyping method suitable for the workflow of a diagnostic laboratory. The established method is robust and stable, and it utilizes a one-step reverse transcription and PCR amplification of the 5' untranslated region (5'UTR) and partial Core region of the HCV genome. Amplification products are sequenced using the standard Sanger method, and the genotype is determined by using a freely accessible web-based genotyping tool. The method was validated at the Helsinki University Hospital Laboratory using 238 previously genotyped serum samples. •A new one-step RT-PCR method for the amplification of the 5' untranslated region and partial Core region of hepatitis C virus was established.•HCV genotype is determined using Sanger sequencing and a freely accessible, easy-to-use web-based genotyping tool.•The method is robust, reproducible and suitable for diagnostic laboratory workflow, and it requires no costly instrumentation or specialized sequence analysis skills.
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http://dx.doi.org/10.1016/j.mex.2018.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6060086PMC
April 2018

BK polyomavirus microRNA expression and sequence variation in polyomavirus-associated nephropathy.

J Clin Virol 2018 05 12;102:70-76. Epub 2018 Feb 12.

Department of Virology, Helsinki University Hospital Laboratory and University of Helsinki, 00014 Helsinki, Finland.

Background: BK polyomavirus (BKPyV) infection is a common asymptomatic viral infection in the general population. Severe complications are seen in immunocompromised individuals, such as polyomavirus-associated nephropathy (PyVAN) in renal transplant recipients. Information on BKPyV microRNA expressions is scarce, although polyomavirus-encoded microRNAs have been shown to control viral replication and assist in immune evasion. Whereas the pathogenic role of rearrangements in JC polyomavirus has been well established, little is known about BKPyV rearrangements in PyVAN.

Objectives: To assess viral microRNA expression and transcriptional control region (TCR) sequence variation in PyVAN patients.

Study Design: bkv-miR-B1-3p and bkv-miR-B1-5p microRNA expression was quantified in 55 plasma samples from 9 PyVAN patients and 2 controls using specific miRNA assays. TCR architectures among the viral populations in each patient were characterized by massive parallel sequencing.

Results: bkv-miR-B1-3p and bkv-miR-B1-5p miRNA expression was established in 85.5% and 98.2% of samples, respectively. On average, an 8.9-fold (bkv-miR-B1-3p) and 8.7-fold (bkv-miR-B1-5p) higher expression levels were detected in PyVAN patients as compared to controls. Rearranged BKPyV strains with duplications and deletions were detected in 7/9 PyVAN patients, but 77.6-99.9% of all sequence reads in all samples represented archetype strains.

Conclusions: The frequent detection and increased expression of miRNAs suggest involvement in PyVAN pathogenesis. Despite the predominance of archetype BKPyV strains, the frequent detection of minor rearranged viral populations urges further study on their role in severe kidney disease. Our results suggest that miRNA expression is increased in PyVAN patients, as well as in the presence of rearranged viral strains.
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http://dx.doi.org/10.1016/j.jcv.2018.02.007DOI Listing
May 2018

Archetype JC Polyomavirus (JCPyV) Prevails in a Rare Case of JCPyV Nephropathy and in Stable Renal Transplant Recipients With JCPyV Viruria.

J Infect Dis 2017 11;216(8):981-989

Department of Virology, Helsinki University Hospital Laboratory and University of Helsinki.

Background: JC polyomavirus (JCPyV) is reactivated in approximately 20% of renal transplant recipients, and it may rarely cause JCPyV-associated nephropathy (JCPyVAN). Whereas progressive multifocal leukoencephalopathy of the brain is caused by rearranged neurotropic JCPyV, little is known about viral sequence variation in JCPyVAN owing to the rarity of this condition.

Methods: Using single-molecule real-time sequencing, characterization of full-length JCPyV genomes in urine and plasma samples from 1 patient with JCPyVAN and 20 stable renal transplant recipients with JCPyV viruria was attempted. Sequence analysis of JCPyV strains was performed, with emphasis on the noncoding control region, the major capsid protein gene VP1, and the large T antigen gene.

Results: Exclusively archetype strains were identified in urine from the patient with JCPyVAN. Full-length JCPyV sequences were not retrieved from plasma. Archetype strains were found in urine samples from 19 stable renal transplant recipients, with JCPyV quasispecies detected in 5 samples. In a patient with minor graft dysfunction, a strain with an archetype-like noncoding cont rol region was discovered. Individual point mutations were detected in both VP1 and large T antigen genes.

Conclusions: Archetype JCPyV was dominant in the patient with JCPyVAN and in stable renal transplant recipients. Archetype rather than rearranged JCPyV seems to drive the pathogenesis of JCPyVAN.
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http://dx.doi.org/10.1093/infdis/jix435DOI Listing
November 2017

Prevalence of high-risk human papillomavirus infection and cancer gene mutations in nonmalignant tonsils.

Oral Oncol 2017 10 18;73:77-82. Epub 2017 Aug 18.

Department of Otorhinolaryngology - Head and Neck Surgery, University of Helsinki and Helsinki University Hospital, P.O. Box 263, 00130 Helsinki, Finland.

Objectives: To analyze the prevalence of high-risk HPV (human papillomavirus) and genetic alterations in nonmalignant tonsils.

Methods: We collected benign fresh tonsillar tissue specimens from 477 patients undergoing tonsillectomy because of chronic tonsillitis or tonsillar hypertrophy in 2012 (Group A, n=237) and in 2015 (Group B, n=240). Luminex xMAP technique served to detect E6/E7 DNA from 16 different high-risk HPV types. Tonsillar DNA and peripheral blood leukocyte DNA from the infected individuals were analyzed using Nimblegen SeqCap EZ Comprehensive Cancer Design panel. The panel targets 578 different genes that are relevant in carcinogenesis. HPV negative tonsillar specimens from age- and gender matched individuals were used as controls. All specimens harboring high-risk HPV were analyzed using fluorescence in situ hybridization (FISH).

Results: Five of 477 (1.0%) patients tested positive for the following HPV types: HPV16 (two cases), HPV52 (one case), HPV66 (one case), HPV52 and HPV68 (coinfection, one case). FISH analyses showed that the appearance of HPV in specimens infected with HPV 16 was episomal. Benign tonsils infected with high-risk HPV harbored mutations in EP300, NF1, PIK3CA, and RB1 which are considered relevant in the development of HPV-associated head and neck squamous cell carcinoma (SCC).

Conclusions: The prevalence of high-risk HPV in nonmalignant tonsils is low. High-risk HPV positive tonsils harbored mutations in genes that are commonly altered in HPV-associated head and neck SCC. The role of these mutations in tonsillar carcinogenesis is an interesting target for future research.
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http://dx.doi.org/10.1016/j.oraloncology.2017.08.010DOI Listing
October 2017

JCPyV microRNA in plasma inversely correlates with JCPyV seropositivity among long-term natalizumab-treated relapsing-remitting multiple sclerosis patients.

J Neurovirol 2017 10 22;23(5):734-741. Epub 2017 Aug 22.

Department of Virology, Medicum, University of Helsinki and Helsinki University Hospital Laboratory, Helsinki, Finland.

Sensitive biomarkers are needed to better manage multiple sclerosis (MS) patients for natalizumab (NTZ)-associated risk of progressive multifocal leukoencephalopathy (PML). A currently used risk stratification algorithm, mainly based on JC polyomavirus (JCPyV) serology, has not led to a reduction of PML incidence. Therefore, this study was designed to evaluate the presence and prevalence of JCPyV miRNAs in plasma of NTZ-treated MS patients, and to explore their biomarker potential for NTZ-associated PML risk assessment. Altogether, 102 plasma samples from 49 NTZ-treated and 28 interferon-beta (IFN-β)-treated relapsing-remitting MS patients, and 25 healthy controls (HCs) were analyzed for jcv-miR-J1-5p (5p miRNA) and jcv-miR-J1-3p (3p miRNA) expression. The overall detection rate of 5p miRNA was 84% (41/49) among NTZ-treated patients, 75% (21/28) among IFN-β-treated patients, and 92% (23/25) in HCs. Relative 5p miRNA expression levels were lower in NTZ-treated patients as compared to patients treated with IFN-β (p = 0.027) but not to HCs. Moreover, 5p miRNA expression inversely correlated with anti-JCPyV antibody index among JCPyV seropositive long-term NTZ-treated patients (r = -0.756; p = 0.002). The overall detection rate of 3p miRNA was low. Our results suggest that JCPyV miRNA in plasma may be linked to the reactivation of persistent JCPyV, to enhanced virus replication, and eventually to the risk of developing PML among NTZ-treated MS patients. However, further study is warranted in a larger data set including samples from PML patients to confirm the clinical relevance of JCPyV miRNA as a sign of/in viral reactivation, and to identify its potential to predict developing PML risk.
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http://dx.doi.org/10.1007/s13365-017-0560-xDOI Listing
October 2017

Testing for high-risk HPV in cervical and tonsillar paraffin-embedded tissue using a cartridge-based assay.

APMIS 2017 Oct 24;125(10):910-915. Epub 2017 Jul 24.

Department of Virology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.

This study evaluates the suitability of Xpert HPV (Cepheid, Sunnyvale, CA, USA) test for cervical and tonsillar formalin-fixed paraffin-embedded (FFPE) tissue samples as compared to the tests currently used in diagnostics. Cervical biopsies and liquid cytology (LC) samples were collected from 48 women attending colposcopy. Biopsies were processed for histology and tested for hrHPV using Xpert HPV. LC samples were tested using Xpert and Hybrid Capture 2 (HC2; Qiagen, Hilden, Germany) tests. Also 29 archived tonsillar carcinoma samples were tested using Xpert, and the results were compared with histology and immunohistochemical p16INK4a (p16) staining. Among valid cervical LC samples 46.8% were hrHPV positive using Xpert test and 55.3% with HC2. The sensitivity of Xpert was 84.6% as compared to HC2, and overall test concordance was 91.5%. Test concordance between valid Xpert results from biopsies and LC samples was 84.6%. Among valid tonsillar samples 70.4% were hrHPV positive, and concordance of 96.3% was found between Xpert and p16 staining. To conclude, Xpert HPV test cartridge provides a convenient platform to test individual samples, including FFPE samples. Further studies are needed to establish whether test sensitivity is sufficient to reliably differentiate between hrHPV positive and hrHPV negative head and neck carcinomas.
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http://dx.doi.org/10.1111/apm.12727DOI Listing
October 2017

Human papillomavirus type 8 E7 protein binds nuclear myosin 1c and downregulates the expression of pre-rRNA.

Virus Genes 2017 Dec 21;53(6):807-813. Epub 2017 Jul 21.

Medicum, Department of Virology, University of Helsinki and Helsinki University Hospital, POB 21, 00014, Helsinki, Finland.

Our aim was to search for new cellular binding partners for the E6 and E7 oncogenes of beta human papillomaviruses (HPV), whose direct role in skin carcinogenesis has not been thoroughly investigated. By employing glutathione S-transferase pulldown and coimmunoprecipitation, we identified nuclear myosin 1c as a binding partner of HPV 8 E7 protein. As nuclear myosin 1c is an essential component of the RNA polymerase I transcription complex, we studied the effects of HPV 8 E7 protein expression on ribosomal RNA (rRNA) expression. Here we show that the activity of RNA polymerase I is decreased and that pre-rRNA expression is consequently reduced due to HPV 8 E7 expression. However, the expression levels of mature cytoplasmic 18S and 28S rRNA are retained. We propose that by relieving their resources from the energy-consuming process of rRNA transcription, HPV 8 E7 expressing cells might support more efficient virus replication in the differentiating epithelium.
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http://dx.doi.org/10.1007/s11262-017-1491-6DOI Listing
December 2017

Single-Molecule Sequencing Revealing the Presence of Distinct JC Polyomavirus Populations in Patients With Progressive Multifocal Leukoencephalopathy.

J Infect Dis 2017 03;215(6):889-895

Department of Virology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.

Background: Progressive multifocal leukoencephalopathy (PML) is a fatal disease caused by reactivation of JC polyomavirus (JCPyV) in immunosuppressed individuals and lytic infection by neurotropic JCPyV in glial cells. The exact content of neurotropic mutations within individual JCPyV strains has not been studied to our knowledge.

Methods: We exploited the capacity of single-molecule real-time sequencing technology to determine the sequence of complete JCPyV genomes in single reads. The method was used to precisely characterize individual neurotropic JCPyV strains of 3 patients with PML without the bias caused by assembly of short sequence reads.

Results: In the cerebrospinal fluid sample of a 73-year-old woman with rapid PML onset, 3 distinct JCPyV populations could be identified. All viral populations were characterized by rearrangements within the noncoding regulatory region (NCCR) and 1 point mutation, S267L in the VP1 gene, suggestive of neurotropic strains. One patient with PML had a single neurotropic strain with rearranged NCCR, and 1 patient had a single strain with small NCCR alterations.

Conclusions: We report here, for the first time, full characterization of individual neurotropic JCPyV strains in the cerebrospinal fluid of patients with PML. It remains to be established whether PML pathogenesis is driven by one or several neurotropic strains in an individual.
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http://dx.doi.org/10.1093/infdis/jiw399DOI Listing
March 2017

Performance of mRNA- and DNA-based high-risk human papillomavirus assays in detection of high-grade cervical lesions.

Acta Obstet Gynecol Scand 2017 Jan 9;96(1):61-68. Epub 2016 Nov 9.

Department of Virology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.

Introduction: The aim was to assess the performance of two commercial assays for the detection of high-risk human papillomavirus (hrHPV): Aptima HPV Assay (Hologic, Inc., Marlborough, MA, USA) which detects mRNA of 14 different hrHPV types, and Hybrid Capture 2 HPV DNA test (HC2; Qiagen, Gaithersburg, MD, USA), which detects the DNA of 13 different hrHPV types. Test performance was compared in the settings of a standard colposcopy clinic, among the regular patient flow.

Material And Methods: Two separate cervical cell samples for Aptima and HC2 testing were collected from women referred to colposcopy or a cervical follow-up visit. Altogether, 481 paired samples were analyzed and all positive samples were also tested using the Aptima HPV 16 18/45 Genotype Assay. Results from the two assays were compared directly and with stratification by histology and cytology from the same sampling visit.

Results: The overall agreement between HC2 and Aptima assays was 92.9% (Kappa coefficient of 0.855). The sensitivity and specificity of the assays in detecting CIN2 were 92.5 and 58.2% for HC2, and 94.0 and 59.3% for Aptima, respectively. No significant differences between the assays were found (p-values >0.5). Both assays detected all CIN3 (n = 30) and carcinoma (n = 2) cases.

Conclusions: The mRNA-based Aptima assay and the extensively studied DNA-based HC2 test performed equally well in detecting high-grade cervical lesions. Our data contribute to the growing evidence base indicating that the mRNA-based Aptima assay could be used for the triage of patients with HPV-associated cervical disease.
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http://dx.doi.org/10.1111/aogs.13041DOI Listing
January 2017

High-level JCPyV viruria after kidney transplantation-Clinical and histopathological findings.

J Clin Virol 2016 12 2;85:75-79. Epub 2016 Nov 2.

Department of Virology, University of Helsinki and Helsinki University Hospital (HUSLAB), PO Box 400, FI 00029 HUS, Helsinki, Finland.

Background: The significance of JC polyomavirus (JCPyV) after kidney transplantation ranges from irrelevant to full-blown nephropathy or PML.

Objectives: To investigate the clinical significance of high-level JCPyV viruria and JCPyV primary infections after kidney transplantation.

Study Design: JCPyV viruria was detected in routine screening by quantitative real-time PCR in 40/238 kidney transplant recipients and was high-level (>10 copies/ml) in 17 patients. A protocol biopsy at the time of JCPyV viruria was available from 10 patients.

Results: Peak urine viral loads were 1.0×10-2.5×10 copies/ml in the 17 high-level viruria patients. 6/15 (40%) patients with high-level JCPyV viruria with pretransplant sera available were JCPyV IgG negative suggesting that JCPyV viruria resulted from the donor graft in most cases. No acute graft dysfunction was associated with JCPyV viruria. No positive SV40 staining was detected in protocol biopsies, and no specific histopathology was associated with high-level viruria; JCPyV nephropathy was not found. No differences were seen in histopathology or graft function at 3 years in patients with high-level viruria compared to non-JCPyV viruric patients transplanted during the same time period, and outcome was similar in patients with presumably primary and reactivated JCPyV. The mean estimated GFR at last follow-up was 44ml/min (range 12-60ml/min). One graft with high-level viruria was lost 9 years posttransplant due to recurrent IgA nephropathy CONCLUSIONS: High-level JCPyV viruria seems to be associated with primary JCPyV infection reflecting the average seroprevalence of 60%, but is not stringently associated with inferior graft function or survival, or histopathological changes.
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http://dx.doi.org/10.1016/j.jcv.2016.10.018DOI Listing
December 2016

Low expression levels of putative HPV encoded microRNAs in cervical samples.

Springerplus 2016 22;5(1):1856. Epub 2016 Oct 22.

Department of Virology, University of Helsinki and Helsinki University Hospital, POB 21, 00014 Helsinki, Finland.

Using small RNA sequencing of libraries established from cervical samples and cervical cancer cell lines, we have previously reported identification of nine and validation of five putative microRNA species encoded by human papillomaviruses (HPV) including five microRNAs encoded by HPV 16. Here we have studied the expression of HPV 16 encoded microRNAs in cervical samples and in HPV 16 containing cell lines. Different sample matrices were collected for the study: 20 paraffin embedded cervical tissue samples, 16 liquid cytology samples, and 16 cervical cell samples from women attending colposcopy due to cervical abnormalities, as well as four HPV 16 containing cell lines. Total RNA was extracted, the samples were spiked with small synthetic control RNAs, and the expression of five HPV 16 encoded microRNAs was assessed by real-time PCR amplification. HPV encoded microRNAs could be frequently detected, albeit at high cycle counts. HPV16-miR-H1 was detected in 3.6 %, HPV16-miR-H3 in 23.6 %, HPV16-miR-H5 in 7.3 %, and HPV16-miR-H6 in 18.2 % of all valid samples. True positive signals for HPV16-miR-H2 could not be detected in any of the samples. Viral microRNAs were detected most frequently in paraffin-embedded samples: in one sample representing normal squamous epithelium, in one cervical intraepithelial neoplasia (CIN) grade 1, one CIN2, three CIN3, two squamous cell carcinoma, three adenocarcinoma in situ, and two adenocarcinoma samples. One liquid cytology sample from a patient with CIN3 as well as all four cell lines were positive for HPV16-miR-H3. In all cases HPV encoded microRNAs were expressed at low levels.
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http://dx.doi.org/10.1186/s40064-016-3524-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5075338PMC
October 2016

Diagnostic and Prognostic Value of MicroRNA in Viral Diseases.

Authors:
Eeva Auvinen

Mol Diagn Ther 2017 02;21(1):45-57

Department of Virology, University of Helsinki and Helsinki University Hospital, Haartmaninkatu 3, POB 21, 00014, Helsinki, Finland.

Virology is probably the most rapidly developing field within clinical laboratory medicine. Adequate diagnostic methods exist for the diagnostics of most acute viral infections. However, emergence of pathogenic viruses or virus strains and new disease associations of known viruses require the establishment of new diagnostic methods, sometimes very rapidly. In the field of chronic or persistent viral diseases, particularly those involving potential of malignant or fatal development, there is a constant need for improved differential diagnostics, monitoring, prognosis and risk assessment. Increasing understanding of disease pathogenesis also enables better patient management and personalized medicine, where companion diagnostics can offer precise and specific tools for individual care. Very often the new tools are offered by molecular diagnostic techniques, and this includes the detection of microRNAs (miRNAs). miRNAs are small regulatory RNA molecules, which regulate the expression of their target genes. They are encoded both by viruses and their host, and both can target either viral or cellular gene expression. In this review the diagnostic possibilities offered by miRNA will be discussed. The focus will be on selected viral and human miRNAs in viral diseases, and examples of miRNAs of putative diagnostic potential will be presented.
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http://dx.doi.org/10.1007/s40291-016-0236-xDOI Listing
February 2017

[Polvomaviruses as causative agents of diseases].

Duodecim 2016 ;132(5):439-45

The number of polyomaviruses causing infections in humans is as high as thirteen. The BK and JC polyomaviruses and the diseases caused by them are best known. For the present, the Merkel cell polyomavirus is the only human polyomavirus considered to be a causative agent of cancer. Other disease associations of polyomaviruses are also subject to active research. All polyomavirus infections are usually harmless respiratory or intestinal infections of childhood. Polyomaviruses, remain in the body for the rest of life, i.e. they persist as part of the body microbiome. Upon weakening of cell-mediated immunity they can also become reactivated and cause clinical problems.
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May 2016

Simultaneous BK Polyomavirus (BKPyV)-associated nephropathy and hemorrhagic cystitis after living donor kidney transplantation.

J Clin Virol 2016 Mar 30;76:4-7. Epub 2015 Dec 30.

Department of Virology, University of Helsinki and Helsinki University Hospital (HUSLAB), PO Box 400, FI 00029HUS, Helsinki, Finland.

BK polyomavirus (BKPyV) commonly reactivates after kidney transplantation, and can cause polyomavirus-associated nephropathy (PyVAN), whereas after allogeneic stem cell transplantation the most frequent manifestation of BKPyV is polyomavirus-associated hemorrhagic cystitis (PyVHC). Despite high-level BKPyV replication in both, the pathogenesis and manifestation of both BKPyV entities appears to differ substantially. We describe an unusual case of simultaneous PyVAN and PyVHC presenting with acute symptoms in a BKPyV-IgG positive recipient eight months after kidney transplantation from a haploidentical living donor, who was BKPyV-IgG negative. Symptoms of cystitis and viremia subsided rapidly after reduction of immunosuppression.
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http://dx.doi.org/10.1016/j.jcv.2015.12.008DOI Listing
March 2016

Individual and Complementary Effects of Human Papillomavirus Oncogenes on Epithelial Cell Proliferation and Differentiation.

Cells Tissues Organs 2016 5;201(2):97-108. Epub 2015 Dec 5.

Research Program in Infection and Cancer, Deutsches Krebsforschungszentrum, Heidelberg, Germany.

Previous studies on human papillomavirus (HPV) type 16 protein functions have established the oncogenic nature of three viral proteins: E5, E6 and E7. Here we have studied the functions of these proteins by functional deletion of the individual E5, E6 or E7, or both E6 and E7 oncogenes in the context of the whole viral genome. These mutants, or the intact wild-type genome, were expressed from the natural viral promoters along with differentiation of epithelial HaCaT cells in three-dimensional collagen raft cultures. High episomal viral copy numbers were obtained using a transfection-based loxp-HPV16-eGFP-N1 vector system. All epithelial equivalents carrying the different HPV type 16 genomes showed pronounced hyperplastic and dysplastic morphology. Particularly the E7 oncogene, with contribution of E6, was shown to enhance cell proliferation. Specifically, the crucial role of E7 in HPV-associated hyperproliferation was clearly manifested. Based on morphological characteristics, immunohistochemical staining for differentiation and proliferation markers, and low expression of E1^E4, we propose that our raft culture models produce cervical intraepithelial neoplasia (CIN)1 and CIN2-like tissue. Our experimental setting provides an alternative tool to study concerted functions of HPV proteins in the development of epithelial dysplasia.
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http://dx.doi.org/10.1159/000441716DOI Listing
December 2016

Expression of toll-like receptors in HPV-positive and HPV-negative oropharyngeal squamous cell carcinoma--an in vivo and in vitro study.

Tumour Biol 2015 Sep 5;36(10):7755-64. Epub 2015 May 5.

Department of Pathology and Oral Pathology, University of Helsinki and Helsinki University Hospital, Haartmaninkatu 3, P.O. Box 21, FI-00014, Helsinki, Finland.

The incidence of oropharyngeal squamous cell carcinoma (OPSCC) has increased over the past decades in many western countries. This trend is mainly attributed to the human papillomavirus (HPV). Cancer-related actions of immunological defense systems are being intensively researched. Human toll-like receptors (TLRs) are a family of pattern recognition receptors that participate in the immunological defense against pathogens, but their actions are also linked to cancer. The expression of TLRs in cervical epithelium alters both during the clearance of HPV infection and the HPV-induced neoplasia, but the expression of TLRs has not been studied in OPSCC. Thirty-five paraffin-embedded, formalin-fixed, squamous cell carcinoma tissue specimens were analyzed for TLRs 2, 3, 4, 5, 7, and 9 and HPV and p16 statuses. The TLR 9 expression was lower in HPV-positive tumors compared with HPV-negative tumors. TLR 7 was expressed in all cancer specimens, but elevated expression was evident in HPV and/or p16-positive tumors. The majority of p16-positive tumors did not express TLR 5, whereas its expression was stronger in p16-negative tumors. The results of in vitro analysis of five human OPSCC cell lines and one human oral tongue squamous cell carcinoma cell line agree with the in vivo trends: low levels of TLR 5 and high levels of TLR 7 in p16-positive OPSCC. Overall, TLR 7 and 9 expression patterns are demonstrated here to relate to the HPV status in vivo and TLR 5 and 7 expression patterns to the p16 status in vivo and in vitro.
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http://dx.doi.org/10.1007/s13277-015-3494-zDOI Listing
September 2015

Expression of BKV and JCV encoded microRNA in human cerebrospinal fluid, plasma and urine.

J Clin Virol 2015 Apr 28;65:1-5. Epub 2015 Jan 28.

Virology, University of Helsinki and Helsinki, University Hospital, 00014 Helsinki, Finland. Electronic address:

Background: BK and JC polyomaviruses encode microRNAs which may facilitate the establishment of persistent infection. MicroRNAs contribute to disease pathogenesis, and may provide useful tools in laboratory diagnostics and patient management.

Objectives: In this pilot work we studied whether viral and cellular microRNAs can be extracted and detected from body fluids to provide added value in a diagnostic laboratory.

Study Design: Altogether 120 human plasma, urine, and cerebrospinal fluid samples from individuals diagnosed with, or suspected of, a severe polyomavirus associated disease, were included in the study. The samples were spiked with unrelated synthetic microRNA to control for sample quality and inhibition. BKV specific bkv-miR-B1-5p, JCV specific jcv-miR-J1-5p, and bkv-miR-B1-3p/jcv-miR-J1-3p, sharing identical sequences between the two viruses, were amplified from human samples using specific TaqMan assays. Expression of 84 circulating human microRNAs was studied in four selected plasma samples in microarray.

Results: jcv-miR-J1-5p and bkv-miR-B1-3p/jcv-miR-J1-3p were frequently amplified from human plasma, urine, and cerebrospinal fluid samples. bkv-miR-B1-5p was amplified from one-third of the samples, which often contained high viral DNA loads. A microarray screen of human microRNAs in plasma samples suggested regulation of several human microRNA expression in BKV positive vs negative samples.

Conclusions: Viral and cellular microRNAs can be processed and detected from human body fluids. They may prove useful in the diagnosis and management of severe polyomavirus associated diseases, calling for further clinical evaluation.
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http://dx.doi.org/10.1016/j.jcv.2015.01.019DOI Listing
April 2015

Inter- and intralaboratory comparison of JC polyomavirus antibody testing using two different virus-like particle-based assays.

Clin Vaccine Immunol 2014 Nov 24;21(11):1581-8. Epub 2014 Sep 24.

Transplantation and Clinical Virology, Department Biomedicine (Haus Petersplatz), University of Basel, Basel, Switzerland Infectious Diseases and Hospital Epidemiology, University Hospital Basel, Basel, Switzerland Division of Infection Diagnostics, Department Biomedicine (Haus Petersplatz), University of Basel, Switzerland

JC polyomavirus (JCPyV) can cause progressive multifocal leukoencephalopathy (PML), a debilitating, often fatal brain disease in immunocompromised patients. JCPyV-seropositive multiple sclerosis (MS) patients treated with natalizumab have a 2- to 10-fold increased risk of developing PML. Therefore, JCPyV serology has been recommended for PML risk stratification. However, different antibody tests may not be equivalent. To study intra- and interlaboratory variability, sera from 398 healthy blood donors were compared in 4 independent enzyme-linked immunoassay (ELISA) measurements generating >1,592 data points. Three data sets (Basel1, Basel2, and Basel3) used the same basic protocol but different JCPyV virus-like particle (VLP) preparations and introduced normalization to a reference serum. The data sets were also compared with an independent method using biotinylated VLPs (Helsinki1). VLP preadsorption reducing ≥35% activity was used to identify seropositive sera. The results indicated that Basel1, Basel2, Basel3, and Helsinki1 were similar regarding overall data distribution (P = 0.79) and seroprevalence (58.0, 54.5, 54.8, and 53.5%, respectively; P = 0.95). However, intra-assay intralaboratory comparison yielded 3.7% to 12% discordant results, most of which were close to the cutoff (0.080 < optical density [OD] < 0.250) according to Bland-Altman analysis. Introduction of normalization improved overall performance and reduced discordance. The interlaboratory interassay comparison between Basel3 and Helsinki1 revealed only 15 discordant results, 14 (93%) of which were close to the cutoff. Preadsorption identified specificities of 99.44% and 97.78% and sensitivities of 99.54% and 95.87% for Basel3 and Helsinki1, respectively. Thus, normalization to a preferably WHO-approved reference serum, duplicate testing, and preadsorption for samples around the cutoff may be necessary for reliable JCPyV serology and PML risk stratification.
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http://dx.doi.org/10.1128/CVI.00489-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4248761PMC
November 2014

Low expression of nuclear Toll-like receptor 4 in laryngeal papillomas transforming into squamous cell carcinoma.

Otolaryngol Head Neck Surg 2014 Nov 9;151(5):785-90. Epub 2014 Sep 9.

Department of Otorhinolaryngology-Head and Neck Surgery, Helsinki University Hospital, University of Helsinki, Helsinki, Finland.

Objective: The malignant transformation rate of recurrent respiratory papillomatosis (RRP), a disease caused by human papillomavirus (HPV), has varied significantly. Cells of the human immune system express toll-like receptors (TLRs) that recognize particles from viruses and bacteria; TLRs are also present on tumor cells, and down-regulation of TLRs has been shown during the progression of HPV-associated neoplasia. The aim of this study was to determine the malignant transformation rate of laryngeal papillomas (LPs) and analyze the potential of TLR2, TLR4, and TLR9 immunoexpression as indicators of the increased cancer risk.

Study Design: Retrospective case-control study.

Setting: Department of Otorhinolaryngology-Head and Neck Surgery, Helsinki University Hospital, University of Helsinki, Helsinki, Finland.

Subjects And Methods: We reviewed all patients with RRP treated for LPs between 1975 and 2011. Data from the Finnish Cancer Registry confirmed the number of patients diagnosed with laryngeal squamous cell carcinoma (LSCC). Laryngeal tissue specimens from patients developing LSCC were subjected to TLR2, TLR4, and TLR9 immunohistochemistry. Nine patients with RRP without malignant transformation and 19 patients with LSCC without a pre-existing LP served as controls.

Results: Nine of 324 patients (2.8%) with RRP developed LSCC. The intensity of nuclear staining of TLR4 was significantly lower in LPs transforming into LSCC than in LPs without malignant transformation. The expression of cytoplasmic TLR4 in LSCC correlated with tumor grade and T stage. Cytoplasmic TLR9 expression was significantly lower in LPs than in LSCC.

Conclusion: The expression of TLR4 may serve as a predictive marker of malignant transformation in LPs. High immunoexpression of cytoplasmic TLR4 in LSCC was associated with a more aggressive disease.
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http://dx.doi.org/10.1177/0194599814549730DOI Listing
November 2014

Identification and validation of human papillomavirus encoded microRNAs.

PLoS One 2013 30;8(7):e70202. Epub 2013 Jul 30.

Institute of Biotechnology, University of Helsinki, Helsinki, Finland.

We report here identification and validation of the first papillomavirus encoded microRNAs expressed in human cervical lesions and cell lines. We established small RNA libraries from ten human papillomavirus associated cervical lesions including cancer and two human papillomavirus harboring cell lines. These libraries were sequenced using SOLiD 4 technology. We used the sequencing data to predict putative viral microRNAs and discovered nine putative papillomavirus encoded microRNAs. Validation was performed for five candidates, four of which were successfully validated by qPCR from cervical tissue samples and cell lines: two were encoded by HPV 16, one by HPV 38 and one by HPV 68. The expression of HPV 16 microRNAs was further confirmed by in situ hybridization, and colocalization with p16INK4A was established. Prediction of cellular target genes of HPV 16 encoded microRNAs suggests that they may play a role in cell cycle, immune functions, cell adhesion and migration, development, and cancer. Two putative viral target sites for the two validated HPV 16 miRNAs were mapped to the E5 gene, one in the E1 gene, two in the L1 gene and one in the LCR region. This is the first report to show that papillomaviruses encode their own microRNA species. Importantly, microRNAs were found in libraries established from human cervical disease and carcinoma cell lines, and their expression was confirmed in additional tissue samples. To our knowledge, this is also the first paper to use in situ hybridization to show the expression of a viral microRNA in human tissue.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0070202PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3728184PMC
April 2014

BK virus viremia in a well-HLA-matched kidney transplant population mainly on low-dose cyclosporine-based immunosuppression.

Clin Transplant 2012 Nov-Dec;26(6):E596-601. Epub 2012 Oct 22.

Division of Nephrology, Department of Medicine, Helsinki University Central Hospital, University of Helsinki, Helsinki, Finland.

The incidence and clinical course of polyomavirus-associated nephropathy (PyVAN) in our well-HLA-matched kidney transplant population mainly on low-dose cyclosporine-based triple-drug immunosuppression has not been described in detail. We aimed to characterize our patients with PyVAN and BK virus (BKV) viremia. Among 166 kidney transplantations between January 2007 and February 2011 followed up at Helsinki University Hospital nephrology clinic, 136 were screened for BKV viremia by quantitative analysis of BKV DNA in plasma. PyVAN was diagnosed by biopsy histopathology and SV40 T-antigen detection. BKV viremia or PyVAN were treated by reducing immunosuppression. BKV viremia was detected in 12 (9%) patients. PyVAN was diagnosed in six patients (4%). In the six patients with no PyVAN, four had low-level viremia (<10,000 copies/mL) of short duration (<2 months), one had high-level viremia, and one had sustained low-level viremia. After reduction of immunosuppression, all except one patient were able to clear viremia. No grafts were lost due to PyVAN. Even in a low-risk population, BKV viremia and PyVAN occur, highlighting the importance of monitoring viral loads. Reduction of immunosuppression was successful, and no grafts were lost due to PyVAN.
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http://dx.doi.org/10.1111/ctr.12040DOI Listing
May 2013

BK polyomavirus-associated hemorrhagic cystitis among pediatric allogeneic bone marrow transplant recipients: treatment response and evidence for nosocomial transmission.

J Clin Virol 2013 Jan 19;56(1):77-81. Epub 2012 Sep 19.

Division of Hematology-Oncology and Stem Cell Transplantation, Childrens' Hospital and Helsinki University Central Hospital, Helsinki, Finland.

Background: BK polyomavirus-associated hemorrhagic cystitis (BK-PyVHC) is a significant complication of allogenic hematopoietic stem cell transplantation (HSCT), but risk factors and treatment are currently unresolved. BK-PyVHC typically presents with clinical cystitis, macrohematuria, and increasing urine and blood BKV loads.

Objectives: Characterization of children undergoing allogeneic HSCT with BK-PyVHC and their clinical and antibody response to cidofovir treatment.

Study Design: By prospective screening of urine and plasma in 50 pediatric allogenic HSCT performed between 2008 and 2010, we identified 6 (12%) children with BK-PyVHC. Cidofovir was administered intravenously to 5 patients and intravesically to 4 patients (3 double treatments).

Results: Decreasing BKV viremia of>2log(10)copies/mL and clinical resolution was seen in 4 patients over 5-12 weeks. Responses occurred only in patients mounting BKV-specific IgM and IgG responses. Epidemic curve plots, BKV genotyping and contact tracing provided evidence of transmission between 2 BKV-seronegative patients, but ruled out transmission among the remaining four patients

Conclusions: The data suggest that BK-PyVHC may be the result of nosocomial transmission in children with low/undetectable BKV antibodies and raises urgent questions about appropriate infection control measures and the role of cidofovir.
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http://dx.doi.org/10.1016/j.jcv.2012.09.003DOI Listing
January 2013

Cell culture model predicts human disease: Altered expression of junction proteins and matrix metalloproteinases in cervical dysplasia.

BMC Clin Pathol 2012 Aug 3;12. Epub 2012 Aug 3.

Haartman Institute, Department of Virology, University of Helsinki, POB 21 (Haartmaninkatu 3), FIN-00014, Helsinki, Finland.

Background: Cervical cancer is necessarily caused by human papillomaviruses, which encode three oncogenes manifesting their functions by interfering with a number of cellular proteins and pathways: the E5, E6, and E7 proteins. We have earlier found in our microarray studies that the E5 oncogene crucially affects the expression of cellular genes involved in adhesion and motility of epithelial cells.

Methods: In order to biologically validate our previous experimental findings we performed immunohistochemical staining of a representative set of tissue samples from different grades of high-risk human papillomavirus associated cervical disease as well as normal squamous and columnar cervical epithelium. Three-dimensional collagen raft cultures established from E5-expressing and control epithelial cells were also examined. The expression of p16, matrix metalloproteinase (MMP) -7, MMP-16, cytokeratin (CK) 8/18, laminin, E-cadherin and beta-catenin was studied.

Results: In agreement with our previous microarray studies, we found intense staining for E-cadherin and beta-catenin in adherens junctions even in high-grade cervical lesions. Staining for MMP-16 was increased in severe disease as well. No significant change in staining for MMP-7 and cytokeratin 8/18 along with the grade of cervical squamous epithelial disease was observed.

Conclusions: Here we have confirmed, using tissue material from human papillomavirus associated lesions, some of the cellular gene expression modifications that we earlier reported in an experimental system studying specifically the E5 oncogene of papillomaviruses. These findings were partially surprising in the context of cervical carcinogenesis and emphasize that the complexity of carcinogenesis is not yet fully understood. Microarray approaches provide a wide overwiev of gene expression in experimental settings, which may yield biologically valid biomarkers for disease diagnostics, prognosis, and follow-up.
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http://dx.doi.org/10.1186/1472-6890-12-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3495715PMC
August 2012

miRSeqNovel: an R based workflow for analyzing miRNA sequencing data.

Mol Cell Probes 2012 Oct 17;26(5):208-11. Epub 2012 May 17.

DNA Sequencing and Genomics Laboratory, Institute of Biotechnology, University of Helsinki, Helsinki, Finland.

We present miRSeqNovel, an R based workflow for miRNA sequencing data analysis. miRSeqNovel can process both colorspace (SOLiD) and basespace (Illumina/Solexa) data by different mapping algorithms. It finds differentially expressed miRNAs and gives conservative prediction of novel miRNA candidates with customized parameters. miRSeqNovel is freely available at http://sourceforge.net/projects/mirseq/files.
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http://dx.doi.org/10.1016/j.mcp.2012.05.002DOI Listing
October 2012
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