HPV Genome Research Articles

Analysis of genetic variation in human papillomavirus type 16 E1 and E2 in women with cervical infection in Xinjiang, China

BackgroundXinjiang is one of the regions with a high incidence of cervical cancer, and the genetic variation of human papillomavirus may increase its ability to infect the human body and enhance virus-mediated immune escape ability.MethodsSanger sequencing of the HPV16 genome from 165 samples positive for HPV16 infection and phylogenetic analysis of the E1 and E2 genes revealed the gene polymorphism of HPV16 in Xinjiang.ResultsThe results showed that there were 109 samples with variations in HPV16 E1, 48 sites with nucleotide variations (19 missense variations and 29 synonymous variations), and 91 samples with variations in HPV16 E2, 25 sites with nucleotide variations (20 missense variations and five synonymous variations).ConclusionsFrom the phylogenetic tree results, 149 samples were of the European variant and 16 samples were of the Asian variant. No African or North American/Asian variant types were found.

Analysis of the Replication Mechanisms of the Human Papillomavirus Genomes.

The life-cycle of human papillomaviruses (HPVs) includes three distinct phases of the viral genome replication. First, the viral genome is amplified in the infected cells, and this amplification is often accompanied by the oligomerization of the viral genomes. Second stage includes the replication of viral genomes in concert with the host cell genome. The viral genome is further amplified during the third stage of the viral-life cycle, which takes place only in the differentiated keratinocytes. We have previously shown that the HPV18 genomes utilize at least two distinct replication mechanisms during the initial amplification. One of these mechanisms is a well-described bidirectional replication via theta type of replication intermediates. The nature of another replication mechanism utilized by HPV18 involves most likely recombination-dependent replication. In this paper, we show that the usage of different replication mechanisms is a property shared also by other HPV types, namely HPV11 and HPV5. We further show that the emergence of the recombination dependent replication coincides with the oligomerization of the viral genomes and is dependent on the replicative DNA polymerases. We also show that the oligomeric genomes of HPV18 replicate almost exclusively using recombination dependent mechanism, whereas monomeric HPV31 genomes replicate bi-directionally during the maintenance phase of the viral life-cycle.

HPV16-Genotyper: A Computational Tool for Risk-Assessment, Lineage Genotyping and Recombination Detection in HPV16 Sequences, Based on a Large-Scale Evolutionary Analysis

Previous analyses have identified certain but limited evidence of recombination among HPV16 genomes, in accordance with a general perception that DNA viruses do not frequently recombine. In this evolutionary/bioinformatics study we have analyzed more than 3600 publicly available complete and partial HPV16 genomes. By studying the phylogenetic incongruence, similarity plots and the distribution patterns of lineage-specific SNPs, we identify several potential recombination events between the two major HPV16 evolutionary clades. These two clades comprise the (widely considered) phenotypically more benign (lower risk) lineage A and the (widely considered) phenotypically more aggressive (higher risk) non-European lineages B, C and D. We observe a frequency of potential recombinant sequences ranging between 0.3 and 1.2% which is low, but nevertheless considerable. Our findings have clinical implications and highlight that HPV16 genotyping and risk assessment based only on certain genomic regions and not the entire genome may provide a false genotype and, therefore, its associated risk estimate. Finally, based on this analysis, we have developed a bioinformatics tool that automates the entire process of HPV16 lineage genotyping, recombination detection and further identifies, within the submitted sequences, SNPs that have been reported in the literature to increase the risk of cancer.

K-Mer Analyses Reveal Different Evolutionary Histories of Alpha, Beta, and Gamma Papillomaviruses.

Papillomaviruses (PVs) are a heterogeneous group of DNA viruses that can infect fish, birds, reptiles, and mammals. PVs infecting humans (HPVs) phylogenetically cluster into five genera (Alpha-, Beta-, Gamma-, Mu- and Nu-PV), with differences in tissue tropism and carcinogenicity. The evolutionary features associated with the divergence of Papillomaviridae are not well understood. Using a combination of k-mer distributions, genetic metrics, and phylogenetic algorithms, we sought to evaluate the characteristics and differences of Alpha-, Beta- and Gamma-PVs constituting the majority of HPV genomes. A total of 640 PVs including 442 HPV types, 27 non-human primate PV types, and 171 non-primate animal PV types were evaluated. Our analyses revealed the highest genetic diversity amongst Gamma-PVs compared to the Alpha and Beta PVs, suggesting reduced selective pressures on Gamma-PVs. Using a sequence alignment-free trimer (k = 3) phylogeny algorithm, we reconstructed a phylogeny that grouped most HPV types into a monophyletic clade that was further split into three branches similar to alignment-based classifications. Interestingly, a subset of low-risk Alpha HPVs (the species Alpha-2, 3, 4, and 14) split from other HPVs and were clustered with non-human primate PVs. Surprisingly, the trimer-constructed phylogeny grouped the Gamma-6 species types originally isolated from the cervicovaginal region with the main Alpha-HPV clade. These data indicate that characterization of papillomavirus heterogeneity via orthogonal approaches reveals novel insights into the biological understanding of HPV genomes.

APOBEC Mutagenesis Is Concordant between Tumor and Viral Genomes in HPV-Positive Head and Neck Squamous Cell Carcinoma.

APOBEC is a mutagenic source in human papillomavirus (HPV)-mediated malignancies, including HPV+ oropharyngeal squamous cell carcinoma (HPV + OPSCC), and in HPV genomes. It is unknown why APOBEC mutations predominate in HPV + OPSCC, or if the APOBEC-induced mutations observed in both human cancers and HPV genomes are directly linked. We performed sequencing of host somatic exomes, transcriptomes, and HPV16 genomes from 79 HPV + OPSCC samples, quantifying APOBEC mutational burden and activity in both host and virus. APOBEC was the dominant mutational signature in somatic exomes. In viral genomes, there was a mean of five (range 0–29) mutations per genome. The mean of APOBEC mutations in viral genomes was one (range 0–5). Viral APOBEC mutations, compared to non-APOBEC mutations, were more likely to be low-variant allele fraction mutations, suggesting that APOBEC mutagenesis actively occurrs in viral genomes during infection. HPV16 APOBEC-induced mutation patterns in OPSCC were similar to those previously observed in cervical samples. Paired host and viral analyses revealed that APOBEC-enriched tumor samples had higher viral APOBEC mutation rates (p = 0.028), and APOBEC-associated RNA editing (p = 0.008), supporting the concept that APOBEC mutagenesis in host and viral genomes is directly linked and occurrs during infection. Using paired sequencing of host somatic exomes, transcriptomes, and viral genomes, we demonstrated for the first-time definitive evidence of concordance between tumor and viral APOBEC mutagenesis. This finding provides a missing link connecting APOBEC mutagenesis in host and virus and supports a common mechanism driving APOBEC dysregulation.

P–578 Human pappiloma virus 16,18 genome methylation patterns in subfertile women

Abstract Study question A comparison between L1 gene and LCR region methylation status of HPV16 and HPV18 viruses in subfertile women, investigating HPV methylation pattern in cervical cancer and asymptomatic HPV infection. Summary answer CpG methylation was more frequent in L1 gene compared to LCR in both HPV types. Methylation levels were associated with the grade of cervical dysplasia. What is known already HPV infection is a common sexually transmitted disease, related to genital warts and cancer. DNA methylation as a dynamic and strictly controlled process can be involved in numerous cellular processes, cell differentiation, gene expression regulation and genome reprogramming. Human pappiloma virus genome epigenetic alterations may play a key role in HPV life cycle as well as in the oncogenic process in general. However, whether the prevalence of high risk HPV is correlated with female infertility, has yet to be elucidated. Study design, size, duration From January 2015 to December 2019, about 2505 infertile couples were referred to the Human Reproduction Unit of Ioannina University Hospital. A total of 212 clinical and laboratory data from female partners were included in the study. Participants/materials, setting, methods Cervical smears were studied for HPV DNA methylation. CpG methylation was compared among L1 gene and LCR region in both HPV types. A bisulfite modification assay followed by DNA amplification and sequencing was performed to analyse HPV16 and HPV18 genome. Main results and the role of chance In HPV16 types, L1 gene and promoter region indicated high methylation levels in cervical cancer cases. LCR regions methylation levels ranged from 0,5% to 24,2% in asymptomatic HPV16 infection or cervical intraepithelial neoplasia and cervical cancer, respectively. As for L1 gene, the differences between asymptomatic HPV16 infection and cervical cancer cases were statistically significant (P = 0.003). In HPV18 types, L1 gene was methylated in cervical intraepithelial neoplasia and cervical cancer cases. Promoter region methylation levels were high in cervical cancer cases while LCR region methylation levels were low. Limitations, reasons for caution Main limitation is the relatively small size of the collected samples. Wider implications of the findings: HPV genome investigation, as for methylation status, may lead to better understanding and earlier diagnostics of cervical pathology in infertile population. These observations point out the importance of fertility preservation in women at high risk for cervical neoplasia. Trial registration number Not applicable

The application of CRISPR/Cas9 system in cervical carcinogenesis

Integration of high-risk HPV genomes into cellular chromatin has been confirmed to promote cervical carcinogenesis, with HPV16 being the most prevalent high-risk type. Herein, we evaluated the therapeutic effect of the CRISPR/Cas9 system in cervical carcinogenesis, especially for cervical precancerous lesions. In cervical cancer/pre-cancer cell lines, we transfected the HPV16 E7 targeted CRISPR/Cas9, TALEN, ZFN plasmids, respectively. Compared to previous established ZFN and TALEN systems, CRISPR/Cas9 has shown comparable efficiency and specificity in inhibiting cell growth and colony formation and inducing apoptosis in cervical cancer/pre-cancer cell lines, which seemed to be more pronounced in the S12 cell line derived from the low-grade cervical lesion. Furthermore, in xenograft formation assays, CRISPR/Cas9 inhibited tumor formation of the S12 cell line in vivo and affected the corresponding protein expression. In the K14-HPV16 transgenic mice model of HPV-driven spontaneous cervical carcinogenesis, cervical application of CRISPR/Cas9 treatment caused mutations of the E7 gene and restored the expression of RB, E2F1, and CDK2, thereby reversing the cervical carcinogenesis phenotype. In this study, we have demonstrated that CRISPR/Cas9 targeting HPV16 E7 could effectively revert the HPV-related cervical carcinogenesis in vitro, as well as in K14-HPV16 transgenic mice, which has shown great potential in clinical treatment for cervical precancerous lesions.

Nucleosome Positioning on Episomal Human Papillomavirus DNA in Cultured Cells.

Several human papillomaviruses (HPV) are associated with the development of cervical carcinoma. HPV DNA synthesis is increased during the differentiation of infected host keratinocytes as they migrate from the basal layer of the epithelium to the spinous layer, but the molecular mechanism is unclear. Nucleosome positioning affects various cellular processes such as DNA replication and repair by permitting the access of transcription factors to promoters to initiate transcription. In this study, nucleosome positioning on virus chromatin was investigated in normal immortalized keratinocytes (NIKS) stably transfected with HPV16 or HPV18 genomes to determine if there is an association with the viral life cycle. Micrococcal nuclease-treated DNA analyzed by Southern blotting using probes against HPV16 and HPV18 and quantified by nucleosome scanning analysis using real-time PCR revealed mononucleosomal-sized fragments of 140–200 base pairs that varied in their location within the viral genome according to whether the cells were undergoing proliferation or differentiation. Notably, changes in the regions around nucleotide 110 in proliferating and differentiating host cells were common to HPV16 and HPV18. Our findings suggest that changes in nucleosome positions on viral DNA during host cell differentiation is an important regulatory event in the viral life cycle.

Oropharyngeal Squamous Cell Carcinoma Morphology and Subtypes by Human Papillomavirus Type and by 16 Lineages and Sublineages.

Oropharyngeal squamous cell carcinoma (SCC) is increasing in incidence and, in Western countries, strongly associated with transcriptionally-active high-risk human papillomavirus (HPV). Within HPV-positive tumors, there is wide morphologic diversity with numerous histologic subtypes of SCC. There are also variable degrees of keratinization, anaplasia, stromal fibrosis, and maturing squamous differentiation. Unlike in the uterine cervix, where associations between HPV types and lineages/sublineages within types have been investigated with some clear correlations identified, little to no data exists for oropharyngeal SCC. In this study, for a large cohort of oropharyngeal SCC patients, we performed RTPCR for high-risk HPV. For the HPV positive patients, we sequenced the DNA of the entire HPV16 genome and determined lineages and sublineages, correlating HPV status, genotype, and HPV16 lineages/sublineages with SCC subtype and various histologic features. Of the 259 patients, 224 (86.5%) were high-risk HPV positive, of which 210/224 (93.8%) were HPV type 16 and 6/224 (2.7%) HPV type 33. Of the four HPV16 lineages, A was the most frequent (192/214 or 89.8%) and of the HPV16 A sublineages, A1 was the most frequent (112/210 or 53.3%). Patients with HPV negative tumors were more often keratinizing vs other types (23/35 or 65.7%) and thus more likely to have more maturing squamous differentiation and stromal desmoplasia. There was no significant correlation between HPV type (16 versus other), between HPV16 lineage (A versus others), or HPV16 A sublineages (A1 or A2 versus others) and morphologic type of SCC nor the various morphologic features of anaplasia/multinucleation, degree of keratinization, nor amount of stromal desmoplasia. In summary, in our cohort, there was no correlation betweenthe type of HPV, the HPV 16 lineage or sublineage, andany of the histologic features or morphologic SCC subtypes.

Episomal HPV16 responsible for aggressive and deadly metastatic anal squamous cell carcinoma evidenced in peripheral blood

Archival tissue samples collected longitudinally from a patient who died from HPV16-induced high-grade anal intraepithelial squamous cell carcinoma with vertebral HPV16–positive metastasis were retrospectively analyzed by the Capture-HPV method (Capt-HPV) followed by Next-Generation Sequencing (NGS). Full length nucleotide sequences of the same HPV16 were identified from the initial and second anal biopsy samples, from plasma sample and from vertebral metastasis biopsy. Remarkably, HPV was episomal in each sample. The HPV genome sequence was closest to the HPV16 Qv18158E variant subtype (A1 lineage) exhibiting base substitutions and deletions in 7 and 2 HPV loci, respectively. In conclusion, the powerful Capt-HPV followed by NGS allows evidencing the detailed cartography of tumoral and circulating HPV DNA, giving rise to a unique and unexpected episomal virus molecular status in a context of aggressive carcinoma, underlying the importance of HPV status and its association with clinical features for further prospective studies.

Case control study comparing the HPV genome in patients with oral cavity squamous cell carcinoma to normal patients using metagenomic shotgun sequencing

The aim of this study was to carry out a case control study comparing the HPV genome in patients with oral cavity squamous cell carcinoma (OC-SCC) to normal patients using metagenomic shotgun sequencing. We recruited 50 OC-SCC cases which were then matched with a control patient by age, gender, race, smoking status and alcohol status. DNA was extracted from oral wash samples from all patients and whole genome shotgun sequencing performed. The raw sequence data was cleaned, reads aligned with the human genome (GRCH38), nonhuman reads identified and then HPV genotypes identified using HPViewer. In the 50 patients with OC-SCC, the most common subsite was tongue in 26 (52%). All patients were treated with primary resection and neck dissection. All but 2 tumors were negative on p16 immunohistochemistry. There were no statistically significant differences between the cases and controls in terms of gender, age, race/ethnicity, alcohol drinking, and cigarette smoking. There was no statistically significant difference between the cancer samples and control samples in the nonhuman DNA reads (medians 4,228,072 vs. 5,719,715, P value = 0.324). HPV was detected in 5 cases (10%) of OC-SCC (genotypes 10, 16, 98) but only 1 tumor sample (genotype 16) yielded a high number of reads to suggest a role in the etiology of OC-SCC. HPV was detected in 4 control patients (genotypes 16, 22, 76, 200) but all had only 1–2 HPV reads per human genome. Genotypes of HPV are rarely found in patients with oral cancer.

Human Papillomavirus 16 (HPV16) E2 Repression of TWIST1 Transcription Is a Potential Mediator of HPV16 Cancer Outcomes.

Human papillomaviruses (HPVs) are causative agents in around 5% of all cancers, including cervical and oropharyngeal. A feature of HPV cancers is their better clinical outcome compared with non-HPV anatomical counterparts. In turn, the presence of E2 predicts a better clinical outcome in HPV-positive cancers; the reason(s) for the better outcome of E2-positive patients is not fully understood. Previously, we demonstrated that HPV16 E2 regulates host gene transcription that is relevant to the HPV16 life cycle in N/Tert-1 cells. One of the genes repressed by E2 and the entire HPV16 genome in N/Tert-1 cells is TWIST1. Here, we demonstrate that TWIST1 RNA levels are reduced in HPV-positive versus HPV-negative head and neck cancer and that E2 and HPV16 downregulate both TWIST1 RNA and protein in our N/Tert-1 model; E6/E7 cannot repress TWIST1. E2 represses the TWIST1 promoter in transient assays and is localized to the TWIST1 promoter; E2 also induces repressive epigenetic changes on the TWIST1 promoter. TWIST1 is a master transcriptional regulator of the epithelial to mesenchymal transition (EMT), and a high level of TWIST1 is a prognostic marker indicative of poor cancer outcomes. We demonstrate that TWIST1 target genes are also downregulated in E2-positive N/Tert-1 cells and that E2 promotes a failure in wound healing, a phenotype of low TWIST1 levels. We propose that the presence of E2 in HPV-positive tumors leads to TWIST1 repression and that this plays a role in the better clinical response of E2-positive HPV tumors.IMPORTANCE HPV16-positive cancers have a better clinical outcome that their non-HPV anatomical counterparts. Furthermore, the presence of HPV16 E2 RNA predicts a better outcome for HPV16-positive tumors; the reasons for this are not known. Here, we demonstrate that E2 represses expression of the TWIST1 gene; an elevated level of this gene is a marker of poor prognosis for a variety of cancers. We demonstrate that E2 directly binds to the TWIST1 promoter and actively represses transcription. TWIST1 is a master regulator promoting EMT, and here, we demonstrate that the presence of E2 reduces the ability of N/Tert-1 cells to wound heal. Overall, we propose that the E2 repression of TWIST1 may contribute to the better clinical outcome of E2-positive HPV16-positive tumors.

Genetic signatures for lineage/sublineage classification of HPV16, 18, 52 and 58 variants

Increasing evidences indicate that high-risk HPV variants are heterogeneous in carcinogenicity and ethnic dispersion. In this work, we identified genetic signatures for convenient determination of lineage/sublineage of HPV16, 18, 52 and 58 variants. Using publicly available genomes, we found that E2 of HPV16, L2 of HPV18, L1 and LCR of HPV52, and L2, LCR and E1 of HPV58 contain the proper genetic signature for lineage/sublineage classification. Sets of hierarchical signature nucleotide positions were further confirmed for high accuracy (>95%) by classifying HPV genomes obtained from Chinese females, which included 117 HPV16 variants, 48 HPV18 variants, 117 HPV52 variants and 89 HPV58 variants. The circulation of HPV variants posing higher cancer risk in Eastern China, such as HPV16 A4 and HPV58 A3, calls for continuous surveillance in this region. The marker genes and signature nucleotide positions may facilitate cost-effective diagnostic detections of HPV variants in clinical settings.

Targeted next generation sequencing panel for HPV genotyping in cervical cancer

Cervical cancer are generally caused by a persistent infection with the oncogenic virus, HPV. Patients with HPV integration are more prone to develop cervical cancer than patients without integration. In this proof-of-concept study, we aimed to develop a sensitive method based on targeted amplicon based NGS for early and precise detection of high-risk HPV-genotypes that are highly associated with the development of cervical cancer. Furthermore, we aimed to investigate if amplicon based NGS allowed for HPV genotyping in cervical lesions and whether it could detect HPV integration. The cohort included a group of CIN3+ biopsies (n = 64), CIN2 samples that progressed (n = 5), CIN2 samples that regressed (n = 3), healthy controls (n = 10), and plasma samples (n = 10) from cervical cancer patients. Sequencing was performed using a custom targeted NGS panel designed to detect all 25 high-risk and probably high-risk and two low-risk HPV genotypes. The method was validated by the SPF10 PCR-DEIA-LiPA25 assay. In the cohort, the following HPV genotypes were identified: HPV-16, 18, 31, 33, 35, 45, 51, 52, 56, 58, and 59. When comparing the results from the SPF10 PCR-DEIA-LiPA25 analyses with the NGS analyses, there was close to a perfect agreement (K = 0.92) among the genotyped HPV types, while in the two cases with complete disagreement, a third assay was applied, and here the results of the NGS analyses were confirmed. Whereas multiple HPV types were detected by the SPF10 PCR-DEIA-LiPA25 assay, the NGS analysis clearly suggest that there is one predomentant HPV type. The NGS assay was capable of detecting HPV-16 in a previous false-negative sample classified by the INNO-LiPA assay, emphasizing the importance of including multiple regions of the HPV genome when genotyping. For the 10 plasma samples, our NGS analyses showed full agreement with the digital droplet PCR (ddPCR) analyses of HPV positive as well as negative plasma samples. Lastly, the custom panel was capable of detecting the integration of HPV-16 in the SiHa cell line. The HPV panel provides a highly cost-effective method for HPV detection and genotyping, as exemplified by a list price of around 75 € per sample. In conclusion, the current study demonstrates that targeted NGS is capable of detecting and genotyping HPV in both FFPE biopsies and plasma samples. This method provides for early diagnosis and prognosis of cervical cancer disease progression, thereby optimizing the potential of recovery and survival for these patients.

An Exonuclease V–qPCR Assay to Analyze the State of the Human Papillomavirus 16 Genome in Cell Lines and Tissues

Integration of the human papillomavirus (HPV) genome into host cell chromosomes has been observed in a majority of HPV-positive cervical cancers and a subset of oral HPV-associated cancers. HPV integration also occurs in long-term cell culture. Screening for HPV integration can be labor intensive and yield results that are difficult to interpret. Here we describe an assay based on exonuclease V (ExoV/RecBCD) and quantitative polymerase chain reaction (qPCR) to determine if samples from cell lines and tissues contain episomal or integrated HPV. This assay can be applied to screen other small DNA viruses with episomal/linear genome configurations in their viral lifecycle and has the potential to be used in clinical settings to define viral genomic conformations associated with disease. © 2020 Wiley Periodicals LLC. Basic Protocol: Exonuclease V genomic DNA digestion and qPCR for detection of HPV16 genome configuration in cells Support Protocol: Exonuclease V analysis of HPV16 genome configuration in tissues Alternate Protocol: Determining HPV integration type or integrity of HPV episome.

Uncovering the Role of the E1 Protein in Different Stages of Human Papillomavirus 18 Genome Replication

The life cycle of human papillomaviruses (HPVs) comprises three distinct phases of DNA replication: initial amplification, maintenance of the genome copy number at a constant level, and vegetative amplification. The viral helicase E1 is one of the factors required for the initiation of HPV genome replication. However, the functions of the E1 protein during other phases of the viral life cycle are largely uncharacterized. Here, we studied the role of the HPV18 E1 helicase in three phases of viral genome replication by downregulating E1 expression using RNA interference or inducing degradation of the E1 protein via inhibition of casein kinase 2α expression or catalytic activity. We generated a novel modified HPV18 genome expressing Nanoluc and tagged E1 and E2 proteins and created several stable HPV18-positive cell lines. We showed that, in contrast to initial amplification of the HPV18 genome, other phases of viral genome replication involve also an E1-independent mechanism. We characterize two distinct populations of HPV18 replicons existing during the maintenance and vegetative amplification phases. We show that a subset of these replicons, including viral genome monomers, replicate in an E1-dependent manner, while some oligomeric forms of the HPV18 genome replicate independently of E1 function.IMPORTANCE Human papillomavirus (HPV) infections pose serious medical problem. To date, there are no HPV-specific antivirals available due to poor understanding of the molecular mechanisms of virus infection cycle. The infection cycle of HPV involves initial amplification of the viral genomes and maintenance of the viral genomes with a constant copy number, followed by another round of viral genome amplification and new viral particle formation. The viral protein E1 is critical for the initial amplification of the viral genome. However, E1 involvement in other phases of the viral life cycle has remained controversial. In the present study, we show that at least two different replication modes of the HPV18 genome are undertaken simultaneously during the maintenance and vegetative amplification phases, i.e., replication of the majority of the HPV18 genome proceeds under the control of the host cell replication machinery without E1 function, whereas a minority of the genome replicates in an E1-dependent manner.

The assessment of a possible link between HPV‐mediated inflammation, apoptosis, and angiogenesis in Prostate cancer

BackgroundThe aim of this study was to determine the presence of HPV in patients with Prostate cancer (PCa) and its possible association with cancer progression. MethodsIn this case-control study, fresh prostate tissues and blood samples were collected from 90 individuals, including 58 cases samples with PCa and 32 non‐malignant prostate tissue samples as a control group. The expression level of viral genes (E2, E6, and E7) and cellular factors including tumor suppressor proteins (Rb and p53), anti-apoptotic mediators (Bcl-2 and survivin), and some mediators involved in inflammation and angiogenesis was evaluated. ResultsThe presence of the HPV genome was identified in 19 out of the 58 cases (32.7%) and five out of the 32 controls (15.6%). However, there was not any statistically significant relationship between the presence of the HPV genome and PCa (OR = 2.63, 95% C.I = 0.89–7.91, P-value = 0.078). Moreover, the HPV high-risk genotypes 16 and 18 were detected in 47.4% and 31.6% of HPV-infected PCa tissues, respectively. The expression level of the tumor suppressor proteins (Rb and p53) significantly decreased in the HPV-infected samples compared to the HPV negative specimens (P-value = 0.01, P-value = 0.01, respectively). However, the expression level of the anti-apoptotic mediators and those involved in angiogenesis and inflammation significantly increased in the HPV-infected PCa group compared to the HPV-negative PCa and control groups (P-value < 0.05, respectively). ConclusionOur study suggests that although it is not definitely known whether HPV causes PCa, this virus probably modulates PCa cell behavior by affecting inflammation, angiogenesis, and apoptosis mechanisms, which, in turn, promotes tumorigenesis.

Decline in circulating viral and human tumor markers after resection of head and neck carcinoma.

DNA sequencing panels can simultaneously quantify human and viral tumor markers in blood. We explored changes in levels of plasma tumor markers following surgical resection of head and neck carcinoma. In preresection and postresection plasmas, targeted DNA sequencing quantified variants in 28 human cancer genes and levels of oncogenic pathogens (human papillomavirus [HPV], Epstein-Barr virus [EBV], Helicobacter pylori) from 21 patients with head and neck squamous cell carcinoma. Preresection, 11 of 21 patients (52%) had detectable tumor markers in plasma, most commonly TP53 mutation or HPV genome. Several days postresection, levels fell to undetectable in 8 of 10 evaluable patients, while two high-stage patients retained circulating tumor markers. Modern sequencing technology can simultaneously quantify human gene variants and oncogenic viral genomes in plasma. Falling levels of cancer-specific markers upon resection can help identify viral and human markers to track at subsequent timepoints as a means to evaluate efficacy of interventions.

Assessing the Efficacy of Human Papillomavirus Disinfection and the Risk of Transmission from Clinical Lesions

<h3>Background</h3> HPV genomes can be detected on medical devices following hospital disinfectant procedures. Recent reports concluded that oncogenic HPVs derived from laboratory tissue-based models are not susceptible to commonly used high-level disinfectants, intensifying concerns that medical instruments may provide transmission of nosocomial HPVs infections. Therefore, we determined the infectious load of HPVs from clinical lesions and investigated the effectiveness of disinfectants on HPV virions derived from laboratory model systems. <h3>Methods</h3> Infectious HPV virions were isolated from cell culture, organotypic epithelial tissue cultures, and mouse xenografts. Clinical samples from recurrent respiratory papillomas (RRPs) and anogenital warts were obtained under IRB approval. The infectivity of HPV virion stocks was measured by the detection of spliced viral E1^E4 mRNAs in infected keratinocytes. Infections were validated by time-dependent detection of E1^E4 mRNAs, resistance to ribonuclease treatment and susceptibility to antibody-mediated neutralization. Our infectivity assay demonstrated a dynamic range of >3-5 log10. Suspension-based disinfection assays employed diluted ortho-phthalaldehyde (OPA) and 0.825% hypochlorite. <h3>Results</h3> In contrast to prior reports, we found that validated HPV virions obtained from a variety of sources were susceptible to a 2.5 to 4 log10 reduction in infectious titer when exposed as directed to OPA or hypochlorite. Some HPV virion stocks failed to meet the infectivity criteria of time-dependent detection of E1^E4 mRNAs, resistance to ribonuclease and susceptibility to antibody-mediated neutralization. Such unvalidated virus stocks are likely to produce spurious results and lead to confounding conclusions. Assessment of HPV infectious titers from clinical lesions suggests that compared to common warts, clinical RRP and anogenital warts have lower levels of virions present at apical surfaces. <h3>Conclusions</h3> We conclude that laboratory sourced HPVs are susceptible to disinfection by OPA and hypochlorite. As the levels of infectious virions recovered from HPV-induced lesions fall below those used in our assays, proper cleaning and disinfection processing is likely to be effective

Adenoviral Vectors Armed with PAPILLOMAVIRUs Oncogene Specific CRISPR/Cas9 Kill Human-Papillomavirus-Induced Cervical Cancer Cells.

Human papillomaviruses (HPV) cause malignant epithelial cancers including cervical carcinoma, non-melanoma skin and head and neck cancer. They drive tumor development through the expression of their oncoproteins E6 and E7. Designer nucleases were shown to be efficient to specifically destroy HPV16 and HPV18 oncogenes to induce cell cycle arrest and apoptosis. Here, we used high-capacity adenoviral vectors (HCAdVs) expressing the complete CRISPR/Cas9 machinery specific for HPV18-E6 or HPV16-E6. Cervical cancer cell lines SiHa and CaSki containing HPV16 and HeLa cells containing HPV18 genomes integrated into the cellular genome, as well as HPV-negative cancer cells were transduced with HPV-type-specific CRISPR-HCAdV. Upon adenoviral delivery, the expression of HPV-type-specific CRISPR/Cas9 resulted in decreased cell viability of HPV-positive cervical cancer cell lines, whereas HPV-negative cells were unaffected. Transduced cervical cancer cells showed increased apoptosis induction and decreased proliferation compared to untreated or HPV negative control cells. This suggests that HCAdV can serve as HPV-specific cancer gene therapeutic agents when armed with HPV-type-specific CRISPR/Cas9. Based on the versatility of the CRISPR/Cas9 system, we anticipate that our approach can contribute to personalized treatment options specific for the respective HPV type present in each individual tumor.