Mateja M. Jelen

Identical Human Papillomavirus (HPV) Genomic Variants Persist in Recurrent Respiratory Papillomatosis for up to 22 Years

Seventy initial and 125 follow-up tissue specimens of laryngeal papillomas, obtained from 70 patients who had had recurrent respiratory papillomatosis for from 1-22 years, were investigated for the presence of human papillomavirus (HPV) DNA and HPV E5a, LCR and/or full-length genomic variants. HPV-6 was found in 130/195, HPV-11 in 63/195, and HPV-6/HPV-11 in 2/195 samples. Within 67/70 (95.7%) patients, all follow-up HPV isolates genetically matched completely initial HPV isolate over the highly variable parts of the genome or over the entire genome. Frequent recurrence of laryngeal papillomas is a consequence of long-term persistence of the identical initial HPV genomic variant.

Serum antibodies to human papillomavirus (HPV) pseudovirions correlate with natural infection for 13 genital HPV types

BACKGROUND Serology for human papillomaviruses (HPV) types -16 and -18 is established as an important tool for studies of HPV vaccinology and epidemiology. However, as there are a large number of oncogenic genital types of HPV there is a need for development of high-throughput, validated HPV serological assays that can be used for more comprehensive seroepidemiological studies and for research on multivalent HPV vaccines. OBJECTIVES To develop a multiplexed pseudovirion-based serological assay (PsV-Luminex) encompassing 21 HPV types and validate the method by correlating the serology with the presence of type specific HPV DNA in cervical samples. STUDY DESIGN Cervical swabs from 3,291 unvaccinated women attending organized cervical screening in Slovenia were tested with 3 different HPV DNA detection methods and presence of HPV DNA compared to presence of serum antibodies to pseudovirions from 15 genital HPV types (HPV-6,-11,-16,-18,-31,-33,-35,-39,-45,-52,-56,-58,-59,-68,-73). RESULTS On average 51% of the HPV DNA positive women were seropositive for the same HPV type that was detected in the cervical specimen. We found a strong correlation with presence of HPV DNA and antibodies to the same HPV type for 13/15 genital HPV types (median OR = 5.7, CI 95% = 2.4-12.9). HPV-52 serology failed the validation and HPV-11 serology could not be validated because only a single woman was positive for HPV-11 DNA. The correlation between serology and HPV DNA status tended to be stronger among women infected with single HPV type (median OR=10.5, CI 95% = 2.4-48.4) than among women with multiple HPV infections (median OR = 4.6, CI 95% = 1.8-11.7). CONCLUSIONS A multiplexed HPV PsV-Luminex assay has been developed and validated to correlate with natural HPV infection for 13 HPV types, thus enabling more comprehensive studies in HPV epidemiology and vaccine research.

Global Genomic Diversity of Human Papillomavirus 11 Based on 433 Isolates and 78 Complete Genome Sequences

ABSTRACT Human papillomavirus type 6 (HPV6) is the major etiological agent of anogenital warts and laryngeal papillomas and has been included in both the quadrivalent and nonavalent prophylactic HPV vaccines. This study investigated the global genomic diversity of HPV6, using 724 isolates and 190 complete genomes from six continents, and the association of HPV6 genomic variants with geographical location, anatomical site of infection/disease, and gender. Initially, a 2,800-bp E5a-E5b-L1-LCR fragment was sequenced from 492/530 (92.8%) HPV6-positive samples collected for this study. Among them, 130 exhibited at least one single nucleotide polymorphism (SNP), indel, or amino acid change in the E5a-E5b-L1-LCR fragment and were sequenced in full. A global alignment and maximum likelihood tree of 190 complete HPV6 genomes (130 fully sequenced in this study and 60 obtained from sequence repositories) revealed two variant lineages, A and B, and five B sublineages: B1, B2, B3, B4, and B5. HPV6 (sub)lineage-specific SNPs and a 960-bp representative region for whole-genome-based phylogenetic clustering within the L2 open reading frame were identified. Multivariate logistic regression analysis revealed that lineage B predominated globally. Sublineage B3 was more common in Africa and North and South America, and lineage A was more common in Asia. Sublineages B1 and B3 were associated with anogenital infections, indicating a potential lesion-specific predilection of some HPV6 sublineages. Females had higher odds for infection with sublineage B3 than males. In conclusion, a global HPV6 phylogenetic analysis revealed the existence of two variant lineages and five sublineages, showing some degree of ethnogeographic, gender, and/or disease predilection in their distribution. IMPORTANCE This study established the largest database of globally circulating HPV6 genomic variants and contributed a total of 130 new, complete HPV6 genome sequences to available sequence repositories. Two HPV6 variant lineages and five sublineages were identified and showed some degree of association with geographical location, anatomical site of infection/disease, and/or gender. We additionally identified several HPV6 lineage- and sublineage-specific SNPs to facilitate the identification of HPV6 variants and determined a representative region within the L2 gene that is suitable for HPV6 whole-genome-based phylogenetic analysis. This study complements and significantly expands the current knowledge of HPV6 genetic diversity and forms a comprehensive basis for future epidemiological, evolutionary, functional, pathogenicity, vaccination, and molecular assay development studies.

Pre-vaccination genomic diversity of human papillomavirus genotype 6 (HPV 6): A comparative analysis of 21 full-length genome sequences

Comparative analysis of 21 full-length genome sequences of human papillomavirus genotype 6 (HPV 6): 18 determined in this study and three sequences available in nucleotide sequence databases, revealed more than 98% nucleotide similarity to the HPV 6 prototype isolate. The minimum and maximum genomic distance between the full-length genomic variants and the prototype sequence was three nucleotide substitutions, and 122 nucleotide substitutions and three insertions, respectively. Detailed sequence analysis of early viral genes E7, E1, E2 and E4, late viral gene L2, and three non-classic non-coding genomic regions (NNCR) revealed the existence of at least four E7, twelve E1, eleven E2, six E4, eleven L2, two NNCR1, two NNCR2, and three NNCR3 genomic variants. In addition, several novel, potentially important amino acid mutations were identified. A phylogenetic tree calculated from viral genome sequences was dichotomic, separating all isolates into HPV 6b (prototypic) and HPV 6a/6vc (non-prototypic) genetic lineages. This study, which contributed the largest number of full-length HPV 6 genome sequences to date, confirmed that HPV 6 diversifies virtually equally across the entire genome by nucleotide (amino acid) exchanges in coding regions and additional nucleotide insertions/deletions in non-coding regions. However, this diversification trend was more evident in non-coding regions LCR and NNCR3 and early viral genes E4, E5a and E5b.

Pre-vaccination seroprevalence of 15 human papillomavirus (HPV) types among women in the population-based Slovenian cervical screening program

OBJECTIVES To estimate seroprevalence of 11 high-risk (hr) HPV types and four low-risk (lr) HPV types among 20-64 years old Slovenian women participating in the population-based cervical cancer screening program. METHODS Serum samples from 3259 women were tested for HPV type-specific antibodies with a multiplexed pseudovirion-based serological assay (PsV-Luminex). RESULTS Seropositivity for any of the 15 HPV types was 65.7%, any of the 11 hr-HPV types 59.2%, and any of the four lr-HPV types 33.1%. Antibodies against at least one of the four vaccine HPV types (HPV 6, 11, 16, 18) were detected in 40.8% women. Among hr-HPV types, seropositivity was highest for HPV 16 (25.2%) and among lr-HPV types for HPV 6 (19.1%). Age-specific HPV16 seropositivity was highest among 30-39 years old (29.6%) and decreased with increasing age to 14.0% among 60-64 years old. CONCLUSION The lifetime sexual exposure to genital HPV types is substantial, emphasising the need for HPV vaccination.

Importance of MicroRNAs in Hepatitis B and C Diagnostics and Treatment

MicroRNAs (miRNAs) are small‐sized RNAs with ability to regulate gene expression and have been recently discovered as promising diagnostic and therapeutic biomarkers in the field of clinical medicine and microbiology, specifically in viral diseases. Infections with hepatitis B virus (HBV) or hepatitis C virus (HCV) often lead to chronic infections and development of liver hepatocellular carcinoma (HCC). Challenges in early diag‐ nosis of HCC and rapid development of novel HCV antivirals call for identification of novel miRNA biomarkers. An extensive selection of single miRNAs and miRNA pan‐ els has been provided by accumulating studies, discovering miRNA potentials in HBV and HCV diagnostics and treatment. Currently, the diagnostic potential of miRNAs in HBV and HCV has not been established yet. However, a promising HCV treatment drug Miravirsen, a locked nucleic acid, complementary to miRNA‐122, has entered a human clinical trial recently. In this review, we outline the role of miRNAs in HBV and HCV pathogenesis and differences in up‐ and downregulation of miRNAs upon HBV and HCV infection and HCC development.

Comparison of SNP Genotypes Related to Proliferative Vitreoretinopathy (PVR) across Slovenian and European Subpopulations

The present study investigated the distribution of genotypes within single nucleotide polymorphisms (SNPs) in genes, related to PVR pathogenesis across European subpopulations. Genotype distributions of 42 SNPs among 96 Slovenian healthy controls were investigated and compared to genotype frequencies in 503 European individuals (Ensembl database) and their subpopulations. Furthermore, a case-control status was simulated to evaluate effects of allele frequency changes on statistically significant results in gene-association studies investigating functional polymorphisms. In addition, 96 healthy controls were investigated within 4 SNPs: rs17561 (IL1A), rs2069763 (IL2), rs2229094 (LTA), and rs1800629 (TNF) in comparison to PVR patients. Significant differences (P < 0.05) in distribution of genotypes among 96 Slovenian participants and a European population were found in 10 SNPs: rs3024498 (IL10), rs315952 (IL1RN), rs2256965 (LST1), rs2256974 (LST1), rs909253 (LTA), rs2857602 (LTA), rs3138045 (NFKB1A), rs3138056 (NFKB1A), rs7656613 (PDGFRA), and rs1891467 (TGFB2), which additionally showed significant differences in genotype distribution among European subpopulations. This analysis also showed statistically significant differences in genotype distributions between healthy controls and PVR patients in rs17561 of the IL1A gene (OR, 3.00; 95% CI, 0.77–11.75; P = 0.036) and in rs1800629 of the TNF gene (OR, 0.48; 95% CI, 0.27–0.87; P = 0.014). Furthermore, we have shown that a small change (0.02) in minor allele frequency (MAF) significantly affects the statistical p value in case-control studies. In conclusion, the study showed differences in genotype distributions in healthy populations across different European countries. Differences in distribution of genotypes may have had influenced failed replication results in previous PVR-related SNP-association studies.

O16.6 Pre-Vaccination Seroprevalence of 15 Human Papillomavirus (HPV) Types Among Slovenian Women Screened For Cervical Cancer

Background Our objective was to estimate the seroprevalence of 11 high-risk (hr) HPV types (HPV 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59) and four low-risk (lr) HPV types (HPV 6, 11, 68, 73) among Slovenian women screened for cervical cancer before the introduction of vaccination against HPV. Methods 3259 serum specimens from the Slovenian HPV prevalence survey were tested for HPV type-specific antibodies with a multiplexed pseudovirion-based serological assay (PsV-Luminex). Results Seropositivity for any of the 15 HPV types was 65.7%, any of the 11 hr-HPV types 59.2%, and any of the four lr-HPV types 33.1%. Antibodies against multiple HPV types were more common (45.3%) than against single HPV types (20.4%). Antibodies against at least one of the four vaccine HPV types (HPV 6, 11, 16, and 18) were detected in 40.8% women. Among hr-HPV types seropositivity was the highest for HPV 16 (25.2%) and among lr-HPV types for HPV 6 (19.1%). Age-specific seropositivity for HPV 16 was the highest among 30–39 years old women (29.6%) and decreased with increasing age to 14.0% among 60–64 years old women (p = 0.014). Seropositivity for any of the hr-HPV among women with pathological cytology was 76.8% and those negative for intraepithelial lesion or malignancy 58.3% (p < 0.001). Conclusion Our results show a substantial burden of lifetime sexual exposure to these 15 HPV types before the introduction of vaccination and also a relatively high cumulative exposure to at least one of the four vaccine HPV types. Thus, vaccination of females before sexual debut with a quadrivalent HPV vaccine has a potential to contribute to a substantial reduction of the burden of cervical infections and cervical cancer as well as some other HPV related morbidity, including genital warts. Our data also present the baseline for monitoring HPV long-term vaccination impact.