Lea Hošnjak

Verrucous carcinoma of the head and neck - not a human papillomavirus-related tumour?

Association between verrucous carcinoma (VC) of the head and neck and human papillomaviruses (HPV) is highly controversial. Previous prevalence studies focused mostly on α‐PV, while little is known about other PV genera. Our aim was to investigate the prevalence of a broad spectrum of HPV in VC of the head and neck using sensitive and specific molecular assays. Formalin‐fixed, paraffin‐embedded samples of 30 VC and 30 location‐matched normal tissue samples were analysed, by using six different polymerase chain reaction‐based methods targeting DNA of at least 87 HPV types from α‐PV, β‐PV, γ‐PV and μ‐PV genera, and immunohistochemistry against p16 protein. α‐PV, γ‐PV and μ‐PV were not detected. β‐PV DNA was detected in 5/30 VC (16.7%) and in 18/30 normal tissue samples (60.0%): HPV‐19, ‐24 and ‐36 were identified in VC, and HPV‐5, ‐9, ‐12, ‐23, ‐24, ‐38, ‐47, ‐49 and ‐96 in normal tissue, whereas HPV type was not determined in 2/5 cases of VC and in 6/18 normal tissue samples. p16 expression was detected in a subset of samples and was higher in VC than in normal tissue. However, the reaction was predominantly cytoplasmic and only occasionally nuclear, and the extent of staining did not exceed 75%. Our results indicate that α‐PV, γ‐PV and μ‐PV are not associated with aetiopathogenesis of VC of the head and neck. β‐PV DNA in a subset of VC and normal tissue might reflect incidental colonization, but its potential biological significance needs further investigation.

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.

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.

Characterization of two novel gammapapillomaviruses, HPV179 and HPV184, isolated from common warts of a renal-transplant recipient.

Gammapapillomavirus (Gamma-PV) is a diverse and rapidly expanding PV-genus, currently consisting of 76 fully characterized human papillomavirus (HPV) types. In this study, DNA genomes of two novel HPV types, HPV179 and HPV184, obtained from two distinct facial verrucae vulgares specimens of a 64 year-old renal-transplant recipient, were fully cloned, sequenced and characterized. HPV179 and HPV184 genomes comprise 7,228-bp and 7,324-bp, respectively, and contain four early (E1, E2, E6 and E7) and two late genes (L1 and L2); the non-coding region is typically positioned between L1 and E6 genes. Phylogenetic analysis of the L1 nucleotide sequence placed both novel types within the Gamma-PV genus: HPV179 was classified as a novel member of species Gamma-15, additionally containing HPV135 and HPV146, while HPV184 was classified as a single member of a novel species Gamma-25. HPV179 and HPV184 type-specific quantitative real-time PCRs were further developed and used in combination with human beta-globin gene quantitative real-time PCR to determine the prevalence and viral load of the novel types in the patient’s facial warts and several follow-up skin specimens, and in a representative collection, a total of 569 samples, of HPV-associated benign and malignant neoplasms, hair follicles and anal and oral mucosa specimens obtained from immunocompetent individuals. HPV179 and HPV184 viral loads in patients’ facial warts were estimated to be 2,463 and 3,200 genome copies per single cell, respectively, suggesting their active role in the development of common warts in organ-transplant recipients. In addition, in this particular patient, both novel types had established a persistent infection of the skin for more than four years. Among immunocompetent individuals, HPV179 was further detected in low-copy numbers in a few skin specimens, indicating its cutaneous tissue tropism, while HPV184 was further detected in low-copy numbers in one mucosal and a few skin specimens, suggesting its dual tissue tropism.

Complete Genome Sequence of a Novel Human Betapapillomavirus, HPV-159

ABSTRACT A novel human papillomavirus (HPV), now officially recognized as HPV-159, isolated from an anal swab, was fully cloned, sequenced, and genetically characterized. HPV-159 has a genomic organization that is typical of cutaneotrophic HPV types, and it belongs to the genus Betapapillomavirus.

Rapid detection and typing of Molluscum contagiosum virus by FRET-based real-time PCR

A fluorescence resonance energy transfer (FRET)-based real-time PCR (RT-PCR) was developed for very sensitive and specific detection of Molluscum contagiosum virus (MCV), as well as reliable differentiation of the two MCV subtype genetic lineages, MCV1 and MCV2, in a single reaction. The assay employs modified primers specific for the viral MC021L gene and uses two novel FRET hybridization probes to detect polymorphisms specific for each of the two subtypes. The sensitivity of the assay at a 95% detection level for both MCV subtypes was 3.3 DNA copies/reaction and the dynamic range was nine orders of magnitude, discriminating 10-10(9) viral genome equivalents/reaction. Post-amplification probe-specific dissociation analysis differentiated the two viral subtypes reliably in all tested concentrations. Testing of 43 tissue specimens clinically diagnosed as MCV lesions showed complete agreement with the results obtained with previously described MCV specific MC080R Taqman RT-PCR and MC021L whole gene sequencing. The novel assay is simple, robust and easy to perform, and may be of great value for clinical and epidemiological studies of MCV infections and related conditions.

Detection of alpha human papillomaviruses in archival formalin-fixed, paraffin-embedded (FFPE) tissue specimens.

Formalin-fixed, paraffin-embedded (FFPE) tissue specimens stored in pathology departments worldwide are an invaluable source for diagnostic purposes when fresh clinical material is unavailable as well as for retrospective molecular and epidemiological studies, especially when dealing with rare clinical conditions for which prospective collection is not feasible. Accurate detection of HPV infection in these specimens is particularly challenging because nucleic acids are often degraded and therefore, not suitable for amplification of larger fragments of the viral genome or viral gene transcripts. This review provides a brief summary of molecular methods for detecting alpha-HPV DNA/RNA in FFPE tissue specimens. We specifically address the key procedural and environmental factors that have the greatest impact on the quality of nucleic acids extracted from FFPE tissue specimens, and describe some solutions that can be used to increase their integrity and/or amplifiability. Moreover, commonly used methods for HPV DNA/RNA detection in FFPE tissue specimens are presented and discussed, focusing on studies using polymerase chain reaction as an HPV detection method and published after 1999. Finally, we briefly summarize our 22 years of experience with HPV detection in FFPE tissue specimens.

Identification of a Novel Human Papillomavirus, Type HPV199, Isolated from a Nasopharynx and Anal Canal, and Complete Genomic Characterization of Papillomavirus Species Gamma-12

The novel human papillomavirus type 199 (HPV199) was initially identified in a nasopharyngeal swab sample obtained from a 25 year-old immunocompetent male. The complete genome of HPV199 is 7,184 bp in length with a GC content of 36.5%. Comparative genomic characterization of HPV199 and its closest relatives showed the classical genomic organization of Gammapapillomaviruses (Gamma-PVs). HPV199 has seven major open reading frames (ORFs), encoding five early (E1, E2, E4, E6, and E7) and two late (L1 and L2) proteins, while lacking the E5 ORF. The long control region (LCR) of 513 bp is located between the L1 and E6 ORFs. Phylogenetic analysis additionally confirmed that HPV-199 clusters into the Gamma-PV genus, species Gamma-12, additionally containing HPV127, HV132, HPV148, HPV165, and three putative HPV types: KC5, CG2 and CG3. HPV199 is most closely related to HPV127 (nucleotide identity 77%). The complete viral genome sequence of additional HPV199 isolate was determined from anal canal swab sample. Two HPV199 complete viral sequences exhibit 99.4% nucleotide identity. To the best of our knowledge, this is the first member of Gamma-PV with complete nucleotide sequences determined from two independent clinical samples. To evaluate the tissue tropism of the novel HPV type, 916 clinical samples were tested using HPV199 type-specific real-time PCR: HPV199 was detected in 2/76 tissue samples of histologically confirmed common warts, 2/108 samples of eyebrow hair follicles, 2/137 anal canal swabs obtained from individuals with clinically evident anal pathology, 4/184 nasopharyngeal swabs and 3/411 cervical swabs obtained from women with normal cervical cytology. Although HPV199 was found in 1.4% of cutaneous and mucosal samples only, it exhibits dual tissue tropism. According to the results of our study and literature data, dual tropism of all Gamma-12 members is highly possible.

Morphological characteristics of conjunctival squamous papillomas in relation to human papillomavirus infection

Objective To determine the prevalence of a broad spectrum of human papillomavirus (HPV) types in conjunctival papillomas and a possible difference in clinical and histopathological presentation of HPV-positive and HPV-negative papillomas. Methods Formalin-fixed, paraffin-embedded papilloma tissue specimens obtained from 25 patients were analysed using six different PCR-based methods targeting 87 HPV types from four different papillomavirus (PV) genera: α-PV, β-PV, γ-PV and µ-PV, and in situ hybridisation for HPV-6/HPV-11. Slides were reviewed for pedunculated or sessile growth, the presence of goblet cells, keratinising or non-keratinising epithelium, elastosis, atypia and koilocytes. Results α-PV types HPV-6 and HPV-11 were detected in 19/25 (76%) conjunctival papilloma tissue specimens, 9 (47%) of which were also HPV-6/HPV-11 positive with in situ hybridisation. Six different β-PV types—HPV-9, HPV-12, HPV-20, HPV-21, HPV-22, HPV-24—were additionally detected in four cases, all of which were also HPV-6/HPV-11 positive. No γ-PVs or µ-PVs were found in any of the tested tissues samples. Extralimbal location (p=0.021), presence of goblet cells (p=0.005), non-keratinising squamous epithelium (p=0.005), and absence of elastosis (p=0.005) were associated with the presence of HPV-6/HPV-11. Conclusions We demonstrated that certain clinical and histological features are more frequently associated with HPV infection and that HPV genera other than α-PV are most probably not significant factors in conjunctival papilloma occurrence.

Novel Human Papillomavirus Type 174 from a Cutaneous Squamous Cell Carcinoma

ABSTRACT We report the cloning and characterization of a novel human papillomavirus (HPV), now officially recognized as HPV-174, isolated from a cutaneous squamous cell carcinoma. HPV-174 has a genome organization characteristic of cutaneous HPV types, clusters to the genus Betapapillomavirus, species β2, and is most closely related to HPV-145.