Differentiation of highly pathogenic strains of human JC polyomavirus in neurological patients by next generation sequencing.

BACKGROUND: JC polyomavirus (JCPyV) persists asymptomatic in more than half of the human population. Immunocompromising conditions may cause reactivation and acquisition of neurotropic rearrangements in the viral genome, especially in the non-coding control region (NCCR). Such rearranged JCPyV strains are strongly associated with the development of progressive multifocal leukoencephalopathy (PML). METHODS: Using next-generation sequencing (NGS) and bioinformatics tools, the NCCR was characterized in cerebrospinal fluid (CSF; N = 21) and brain tissue (N = 16) samples from PML patients (N = 25), urine specimens from systemic lupus erythematosus patients (N = 2), brain tissue samples from control individuals (N = 2) and waste-water samples (N = 5). Quantitative PCR was run in parallel for diagnostic PML samples. RESULTS: Archetype NCCR (i.e. ABCDEF block structure) and archetype-like NCCR harboring minor mutations were detected in two CSF samples and in one CSF sample and in one tissue sample, respectively. Among samples from PML patients, rearranged NCCRs were found in 8 out of 21 CSF samples and in 14 out of 16 brain tissue samples. Complete or partial deletion of the C and D blocks was characteristic of most rearranged JCPyV strains. From ten CSF samples and one tissue sample NCCR could not be amplified. CONCLUSIONS: Rearranged NCCRs are predominant in brain tissue and common in CSF from PML patients. Extremely sensitive detection and identification of neurotropic viral populations in CSF or brain tissue by NGS may contribute to early and accurate diagnosis, timely intervention and improved patient care.

Exploring JC polyomavirus sequences and human gene expression in brain tissue of patients with progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a rare neurological condition associated with reactivation of dormant JC polyomavirus (JCPyV). In this study, we characterized gene expression and JCPyV rearrangements in PML brain tissue. Infection of white matter astrocytes and oligodendrocytes as well as occasional brain cortex neurons was shown. PML brain harbored exclusively rearranged JCPyV variants. Viral transcripts covered the whole genome on both strands. Strong differential expression of human genes associated with neuroinflammation, blood-brain-barrier permeability and neurodegenerative diseases was shown. Pathway analysis revealed wide immune activation in PML brain. The study provides novel insights into the pathogenesis of PML.

JC Polyomavirus Modifies the Expression of Human microRNAs in Progressive Multifocal Leukoencephalopathy Brain.

Progressive multifocal leukoencephalopathy (PML) is a severe neurological condition caused by reactivation of JC polyomavirus (JCPyV) in immunosuppression. Asymptomatic JCPyV persists in peripheral tissues. Upon reactivation, neurotropic rearrangements may emerge, and the virus gains access to the brain. To assess the mechanisms of PML pathogenesis, brain tissue material from PML patients was collected for small RNA sequencing. Upregulation of 8 microRNAs (miRNAs) in PML brain was validated using quantitative microRNA polymerase chain reaction (PCR). Bioinformatics tools were utilized to identify major associations of the upregulated miRNAs: neuroinflammation and blood-brain barrier disruption. The results indicate involvement of human miRNA regulation in PML pathogenesis.

Overtreatment rate after immediate local excision of suspected cervical intraepithelial neoplasia: A prospective cohort study.

OBJECTIVE: The gold standard of cervical intraepithelial neoplasia (CIN) treatment is large loop excision of the transformation zone (LLETZ) after histopathological diagnosis from punch biopsies. In addition, treatment may be appropriate at initial colposcopy. Our objective was to study the applicability of immediate treatment strategy according to clinical parameters. METHODS: We conducted a prospective cohort study among patients referred to colposcopy at Helsinki University Hospital, Finland, between January 2014, and September 2018 (ISRCTN10933736). Patients treated with LLETZ, either after biopsies or immediately at initial colposcopy, were included. The main outcome measure was overtreatment (OT) rate defined as normal or low-grade histopathological findings in LLETZ specimen within both treatment groups. RESULTS: A total of 572 patients treated with LLETZ were included: 360 treated after biopsies and 212 treated immediately at initial colposcopy. When LLETZ was performed immediately after high-grade referral cytology and with colposcopic impression of high-grade disease, the overtreatment (OT) rate was 10.0% (95% CI 9.10 to 17.2), whereas when LLETZ was done after biopsy-confirmed high-grade lesions, the OT rate was 18.9% (95% CI 14.7 to 23.7), resulting in risk difference (RD) -8.91% (95% CI -16.0 to -1.82). Among HPV16/18 positive patients the OT rate was 8.22% (95% CI 3.08 to 17.0) for immediate treatment, resulting in RD of -10.7% (95% CI -18.3 to -3.04) compared to LLETZ after biopsies. CONCLUSIONS: Immediate LLETZ does not result in overtreatment when applied on selected cases, especially after high-grade referral cytology and when high-grade lesion is also colposcopically suspected.

Human papillomavirus self-sampling with mRNA testing benefits routine screening.

High risk human papillomavirus (hrHPV) based screening provides the possibility of vaginal self-sampling as a tool to increase screening attendance. In order to evaluate the impact and feasibility of opt-in self-sampling in the Finnish setting, we invited a randomized population of 5350 women not attending screening after age group invitation or after reminder, to attend HPV self-sampling-based screening in the autumn of 2018 in Helsinki. Out of those, 1282 (24.0%) expressed their interest and ordered the sampling package. Eventually 787 women (14.7% of the total invited population) took part in screening, 770 women by providing a vaginal sample within 2 months from invitation and 17 by providing a pap smear in the laboratory. Self-taken samples were collected in Aptima Multitest vials and tested using the Aptima HPV mRNA assay. A high proportion, 158/770 (20.5%) of the samples were positive in the Aptima HPV assay. One hundred and forty-one samples were further submitted to Aptima HPV Genotyping and extended genotyping by a Luminex based assay. Of those, 23 samples (16.3%) were HPV 16 positive and 7 (5.0%) were positive for HPV 18/45; extended genotyping revealed multiple high-risk and low-risk HPV genotypes. At follow-up seven cases of high-grade squamous intraepithelial lesion (HSIL) were diagnosed, which represents 4.4% of HPV positive women and 0.9% of screened women, whereas the rate was 0.5% in routine screening. Our findings suggest that self-sampling with HPV mRNA testing is a feasible approach to improve screening efficacy in a high-risk population among original nonattendees.

JC polyomavirus DNA detection in clinical practice.

BACKGROUND: There are limited data about the use and clinical value of JC polyomavirus (JCPyV) DNA detection in various clinical indications. METHODS: We reviewed the clinical records of 410 patients from whom cerebrospinal fluid (CSF), plasma, urine, or tissue samples had been collected for JCPyV DNA polymerase chain reaction (PCR) between 2012 and 2018. RESULTS: JCPyV DNA was analyzed in 224 plasma, 190 CSF-, 32 urine and 10 tissue samples. 240 patients had a history of hematopoietic stem cell or solid organ transplantation, 159 had nephrological disease, 90 had hematologic malignancies, 58 had neurological disease, 37 had infectious disease and 23 had AIDS/HIV as underlying disease. Six patients had no underlying disease. The main reasons to take CSF or plasma samples were neurological symptoms of unknown etiology. Most urine samples were taken to monitor kidney transplantation patients. JCPyV DNA PCR contributed to the diagnosis of progressive multifocal leukoencephalopathy in eight patients (2.0%), of which seven had hematologic malignancy as an underlying disease. CONCLUSIONS: JCPyV PCR is most informative among immunosuppressed patients with neurologic symptoms. CSF and brain biopsy are useful when there is clinical suspicion of PML, whereas plasma samples are not useful. The value of plasma samples is a matter of dispute in the screening of JCPyV-associated nephropathy, as BK polyomavirus is the causative agent in most polyomavirus-associated nephropathy cases. JCPyV detection is valuable in case the patient has past, current or planned treatment with immunosuppressive drugs.

HPV-Positive Status Is an Independent Factor Associated With Sinonasal Inverted Papilloma Recurrence.

OBJECTIVES: The present study aimed to evaluate human papillomavirus (HPV) occurrence in sinonasal inverted papilloma (SNIP), and to assess factors associated with HPV positivity and SNIP recurrence. STUDY DESIGN: Prospective study. METHODS: We prospectively collected clinical data and fresh tissue specimens from 90 consecutive patients treated for SNIP at Helsinki University Hospital, between 2015 and 2019. Fourteen patients with recurrent SNIP underwent repeated tumor sampling. All tissue specimens were analyzed for the presence of HPV. Factors associated with SNIP recurrence and HPV positivity were assessed. RESULTS: Among 107 SNIP specimens, 14 (13.1%) were positive for low-risk HPV and 6 (5.6%) were positive for high-risk HPV. HPV positivity was associated with an increased risk of recurrence (P = .004). Smoking was significantly associated with HPV positivity in SNIP (P = .01), but a history of HPV-related diseases or patient sexual habits did not correlate with HPV positivity. The recurrence rate was lower among patients with SNIP that underwent an attachment-oriented resection, compared to patients treated without attachment-oriented resections (78.6% vs 25.8%, P < .001). CONCLUSIONS: The risk of SNIP recurrence was highly associated with 1) HPV positivity and 2) surgery without an attachment-oriented resection. Oncogenic HPV was rare in SNIP. LEVEL OF EVIDENCE: 3 Laryngoscope, 132:1714-1718, 2022.

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

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.

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

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.

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

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.

Progressive Multifocal Leukoencephalopathy: Current Insights.

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.

BetaHPV E6 and E7 colocalize with NuMa in dividing keratinocytes.

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.

BK polyomavirus viremia and antibody responses of pediatric kidney transplant recipients in 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 51 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.

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

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.

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

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.

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

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.

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

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.

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

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-ß)-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-ß-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-ß (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.

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

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.

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

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.