Distribution of human papillomavirus genotypes in women with high-grade cervical intraepithelial lesions and cervical carcinoma and analysis of human papillomavirus-16 genomic variants.

AIM: To analyze the distribution of high-risk human papillomavirus (HR-HPV) genotypes and the diversity of HPV-16 genomic variants in Croatian women with high-grade squamous intraepithelial lesions (HSIL) and cervical carcinoma. METHODS: Tissue biopsy specimens were obtained from 324 women with histopathologically confirmed HSIL or cervical carcinoma, 5 women with low-grade SIL, and 49 women with negative histopathology. HR-HPV DNA was detected with Ampliquality HPV-type nucleic-acid hybridization assay, which identifies 29 different HPV genotypes. HPV-16 genomic variants were analyzed by an in-house sequencing. RESULTS: The most common HPV type in women with HSIL was HPV-16, detected in 127/219 (57.9%) specimens. HPV-16 was also the dominant type in squamous cell cervical carcinoma (46/69 or 66.7%) and in adenocarcinoma (18/36 or 50.0%). Out of 378 patients, 360 had HR-HPV (282 single infections and 79 multiple infections), 3 (0.8%) patients had low-risk HPV, and 15 (4%) tested negative. HPV-16 variants were determined in 130 HPV-16 positive specimens, including 74 HSIL and 46 carcinoma specimens. In HSIL specimens, 41 distinct variants were found, 98.6% belonging to the European branch and 1.4% belonging to the African branch. In cervical carcinoma specimens, 95% isolates grouped in 41 variants belonging to the European branch, one isolate (2.5%) belonged to the North American, and one (2.5%) to the Asian-American branch. CONCLUSION: HPV-16, mainly belonging to the European branch, was the most frequent HPV genotype in women from Croatia with histologically confirmed HSIL and cervical cancer.

Assessing Gammapapillomavirus infections of mucosal epithelia with two broad-spectrum PCR protocols.

BACKGROUND: Human papillomaviruses (HPVs) have been divided into mucosal and cutaneous types according to their primary epithelial tissue tropism. However, recent studies showed the presence of several cutaneous types in mucosal lesions and healthy mucosa from different anatomical sites. METHODS: Here, the HPV prevalence and type-specific distribution were assessed in a variety of mucosal samples from 435 individuals using a combination of two established broad-spectrum primer systems: Gamma-PV PCR and CUT PCR. RESULTS: Overall HPV prevalence in anal canal swabs, cervical cancer biopsies, genital warts and oral swabs was 85, 47, 62 and 4%, respectively. In anal canal swabs, Alpha-PVs were most frequently found (59%), followed by Gamma- (37%) and Beta-PVs (4%). The prevalence and persistence of HPV infection in the anal canal of 226 individuals were further explored. Overall HPV, Gamma-PVs and multiple HPV infections were significantly higher in men vs. women (p = 0.034, p = 0.027 and p = 0.003, respectively); multiple HPV infections were more common in individuals ≤40 years (p = 0.05), and significantly higher prevalence of Gamma-PVs and multiple HPV infections was observed in HIV-1-positive vs. HIV-1-negative individuals (p = 0.003 and p = 0.04, respectively). Out of 21 patients with follow-up anal swabs, only one persistent infection with the same type (HPV58) was detected. CONCLUSIONS: Our findings suggest that Gamma-PVs (except species Gamma-6) are ubiquitous viruses with dual muco-cutaneous tissue tropism. Anal canal Gamma-PV infections may be associated with sexual behavior and the host immune status. This study expands the knowledge on Gamma-PVs' tissue tropism, providing valuable data on the characteristics of HPV infection in the anal canal.

Detection of novel Betapapillomaviruses and Gammapapillomaviruses in eyebrow hair follicles using a single-tube 'hanging droplet' PCR assay with modified pan-PV CODEHOP primers.

A modified pan-PV consensus-degenerate hybrid oligonucleotide primer (CODEHOP) PCR was developed for generic and sensitive detection of a broad-spectrum of human papillomaviruses (HPVs) infecting the cutaneous epithelium. To test the analytical sensitivity of the assay we examined 149 eyebrow hair follicle specimens from immunocompetent male patients. HPV DNA was detected in 60 % (89/149) of analysed eyebrow samples with a total of 48 different HPV sequences, representing 21 previously described HPVs and 27 putative novel HPV types. Evidence for ten novel HPV subtypes and seven viral variants, clustering to three out of five genera containing cutaneous HPVs, was also obtained. Thus, we have shown that the modified pan-PV CODEHOP PCR assay is able to identify multiple HPV types, even from different genera, in the same clinical sample. Overall, these results demonstrate that the pan-PV CODEHOP PCR is an excellent tool for screening and identification of novel cutaneous HPVs, even in samples with low viral loads.

Pre-vaccination prevalence of high-risk human papillomaviruses (HPV) in women from Kosovo and their related sociodemographic characteristics.

OBJECTIVES: Kosovo's current health care system does not support organized nationwide cervical cancer screening and human papillomavirus (HPV) vaccination programs. To date, no reliable data are available on cervical cancer incidence and mortality in Kosovo, or on high-risk HPV (HR-HPV) prevalence and HPV type distribution. Our aim is to determinate the pre-vaccination prevalence and distribution of HR-HPVs and to assesses the associations between sociodemographic characteristics and increased risk of HPV infection in women from Kosovo. MATERIAL AND METHODS: Detection of HR-HPV DNA in cytologically evaluated cervical smears was performed using a clinically validated Abbott RealTime High Risk HPV test, Roche Linear Array HPV Genotyping Test, HPV52 type-specific real-time PCR and an in-house GP5+/GP6+/68 PCR. RESULTS: The crude overall prevalence of any of the HR-HPVs was estimated at 13.1% (26/199; 95% confidence interval (CI): 9.1-18.5%), with HPV16 being the most common type (7/26, 26.9%), followed by HPV31 and HPV51, each detected in 4/26 (15.4%) cervical specimens, HPV18, detected in 3/26 (11.5%) specimens, HPV52 and HPV66, each detected in 2/26 (7.7%) specimens, and HPV33, HPV45, HPV56, and HPV58, each detected in a single (3.9%) specimen. Women over 40 (OR = 0.36), older than 18 at sexual debut (odds ratio (OR) = 0.28), those that had delivered at least one child (OR = 0.32), and those that had a history of pregnancy termination (OR = 0.39) were at lower risk for HPV infection. CONCLUSION: Because more than 70% of cervical precancerous lesions could have been prevented in Kosovo using nationwide HPV-based cervical cancer screening and HPV vaccination, it is of outmost importance to implement both programs in the national health care system as soon as possible.

Natural history of human papillomavirus infection of sun-exposed healthy skin of immunocompetent individuals over three climatic seasons and identification of HPV209, a novel betapapillomavirus.

We present the first longitudinal study reporting the natural history of human papillomavirus (HPV) infection in sun-exposed skin of healthy individuals living in a geographical area in which solar UV radiation is influenced by the ozone content of the atmosphere. During three climatic seasons, skin swab samples were obtained from 78 healthy individuals and the prevalence of cutaneous HPVs was assessed with broad-spectrum FAP and CUT primers and determined at 54, 45 and 47 % in spring, summer and winter, respectively. Frequencies of mixed HPV infections were significantly higher in spring with respect to summer and winter (P=0.02). Seventy-one different HPV types/putative types were identified. While 62 volunteers were HPV-infected in at least one season, 23 had persistent infections. ß-PVs (ß-1) were the most prevalent and persistent. Age was associated with both the infection status (P=0.01) and the type of HPV infection (no infection, indeterminate/transient, persistent P=0.02). The molecular/phylogenetic analysis of the newly identified ß-PV, officially designated as HPV209, showed that the virus has a typical genomic organization of cutaneous HPVs with five early (E6, E7, E1, E2 and E4) and two late genes (L2 and L1), which clusters to the species ß-2. This provides useful data on cutaneous HPV infections in high UV-exposed regions.

Molecular characterization, prevalence and clinical relevance of Phodopus sungorus papillomavirus type 1 (PsuPV1) naturally infecting Siberian hamsters (Phodopus sungorus).

Phodopus sungorus papillomavirus type 1 (PsuPV1), naturally infecting Siberian hamsters (Phodopus sungorus) and clustering in the genus Pipapillomavirus (Pi-PV), is only the second PV type isolated from the subfamily of hamsters. In silico analysis of three independent complete viral genomes obtained from cervical adenocarcinoma, oral squamous cell carcinoma and normal oral mucosa revealed that PsuPV1 encodes characteristic viral proteins (E1, E2, E4, E6, E7, L1 and L2) with conserved functional domains and a highly conserved non-coding region. The overall high prevalence (102/114; 89.5 %) of PsuPV1 infection in normal oral and anogenital mucosa suggests that asymptomatic infection with PsuPV1 is very frequent in healthy Siberian hamsters from an early age onward, and that the virus is often transmitted between both anatomical sites. Using type-specific real-time PCR and chromogenic in situ hybridization, the presence of PsuPV1 was additionally detected in several investigated tumours (cervical adenocarcinoma, cervical adenomyoma, vaginal carcinoma in situ, ovarian granulosa cell tumour, mammary ductal carcinoma, oral fibrosarcoma, hibernoma and squamous cell papilloma) and normal tissues of adult animals. In the tissue sample of the oral squamous cell carcinoma individual, punctuated PsuPV1-specific in situ hybridization spots were detected within the nuclei of infected animal cells, suggesting viral integration into the host genome and a potential etiological association of PsuPV1 with sporadic cases of this neoplasm.

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

UNLABELLED: Human papillomavirus 11 (HPV11) is an etiological agent of anogenital warts and laryngeal papillomas and is included in the 4-valent and 9-valent prophylactic HPV vaccines. We established the largest collection of globally circulating HPV11 isolates to date and examined the genomic diversity of 433 isolates and 78 complete genomes (CGs) from six continents. The genomic variation within the 2,800-bp E5a-E5b-L1-upstream regulatory region was initially studied in 181/207 (87.4%) HPV11 isolates collected for this study. Of these, the CGs of 30 HPV11 variants containing unique single nucleotide polymorphisms (SNPs), indels (insertions or deletions), or amino acid changes were fully sequenced. A maximum likelihood tree based on the global alignment of 78 HPV11 CGs (30 CGs from our study and 48 CGs from GenBank) revealed two HPV11 lineages (lineages A and B) and four sublineages (sublineages A1, A2, A3, and A4). HPV11 (sub)lineage-specific SNPs within the CG were identified, as well as the 208-bp representative region for CG-based phylogenetic clustering within the partial E2 open reading frame and noncoding region 2. Globally, sublineage A2 was the most prevalent, followed by sublineages A1, A3, and A4 and lineage B. IMPORTANCE: This collaborative international study defined the global heterogeneity of HPV11 and established the largest collection of globally circulating HPV11 genomic variants to date. Thirty novel complete HPV11 genomes were determined and submitted to the available sequence repositories. Global phylogenetic analysis revealed two HPV11 variant lineages and four sublineages. The HPV11 (sub)lineage-specific SNPs and the representative region identified within the partial genomic region E2/noncoding region 2 (NCR2) will enable the simpler identification and comparison of HPV11 variants worldwide. This study provides an important knowledge base for HPV11 for future studies in HPV epidemiology, evolution, pathogenicity, prevention, and molecular assay development.

Anal verrucous carcinoma is not related to infection with human papillomaviruses and should be distinguished from giant condyloma (Buschke-Löwenstein tumour).

AIMS: Verrucous carcinoma (VC) is a variant of well-differentiated squamous cell carcinoma and in the anal region is regarded as synonymous with giant condyloma (Buschke-Löwenstein tumour) (BLT). Aetiology, diagnostic criteria and clinical behaviour of both lesions are controversial. Recent studies suggest that VC at other sites is not associated with human papillomaviruses (HPV). We hypothesized that anal VC is also not related to HPV, while BLT is a HPV-induced lesion. METHODS AND RESULTS: Ten cases of VC and four cases of BLT were included. Several techniques were used for HPV detection: in-situ hybridization for HPV6, 11, 16 and 18, six different polymerase chain reaction (PCR) protocols for detection of at least 89 HPV types from alpha-, beta-, gamma- and mu-PV genera and in-situ hybridization for high-risk HPV E6/E7 mRNA; p16 immunohistochemistry and morphometric analysis were also performed. Alpha-, gamma- and mu-PVs were not found in any case of VC, while HPV6 was detected in all cases of BLT. p16 overexpression was not present in any of the lesions. Among microscopic features, only the absence of koilocytosis and enlarged spinous cells seem to be useful to distinguish VC from BLT. CONCLUSIONS: Our results suggest that anal VC, similarly to VC at other sites, is not associated with HPV infection, and must be distinguished from BLT, which is associated with low-risk HPV. Only with well-set diagnostic criteria will it be possible to ascertain clinical behaviour and optimal treatment for both lesions.

The effect of single nucleotide polymorphisms in G-rich regions of high-risk human papillomaviruses on structural diversity of DNA.

BACKGROUND: Infection with high-risk human papillomaviruses (HPVs) can lead to development of cancer of the head and neck and anogenital regions. G-rich sequences found in genomes of high-risk HPVs can fold into non-canonical secondary structures that could serve as 3D motifs distinct from double-stranded DNA and present recognition sites for ligands and opportunity for gene expression modulation. METHODS: Combination of UV, CD and NMR spectroscopy and PAGE electrophoresis were used as they offer complementary insights into structural changes of G-rich oligonucleotides. RESULTS: G-rich region of HPV16 is shown to preferentially form hairpin structures, while regions of HPV18, HPV52 and HPV58 fold into four-stranded DNA structures called G-quadruplexes. Single nucleotide polymorphisms found in G-rich sequences have been found to promote formation of hairpin structures of HPV16 and have affected number of species formed in G-rich region of HPV52, whereas they have exhibited minimal effect on the formation of HPV18 and HPV58 G-quadruplex structures. These structural changes were reflected in differences in apparent thermal stabilities. CONCLUSIONS: Potential of G-rich sequences as drug targets was evaluated based on the results of the current study. HPV16 and HPV18 are considered less appropriate targets due to several single nucleotide polymorphisms and low stability, respectively. On the other hand, HPV52 and HPV58 could be used for small-molecule mediated stabilization. GENERAL SIGNIFICANCE: G-rich sequences occurring in high-risk HPVs can fold into hairpin and G-quadruplex structures that could be potentially utilized as drug targets. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Global genomic diversity of human papillomavirus 6 based on 724 isolates and 190 complete genome sequences.

UNLABELLED: 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.