Diego Chouhy

Differential expression of the human homologue of drosophila discs large oncosuppressor in histologic samples from human papillomavirus–associated lesions as a marker for progression to malignancy

High‐risk HPVs play a causal role in the development of cervical cancer, and their E6 oncoproteins target h‐Dlg for ubiquitin‐mediated proteolysis. The h‐Dlg oncosuppressor is associated with cell–cell interactions, and deregulation of these structures leads to defective cell adhesion, loss of cell polarity and unregulated proliferation. We evaluated the contribution of this E6 activity in the progression to malignancy in HPV infections by analyzing h‐Dlg expression in HPV‐associated lesions. We analyzed h‐Dlg in cervical, laryngeal, vulvar, colon and kidney histologic samples by Dlg immunohistochemistry. HPV association was ascertained by a PCR‐colorimetric method. Although Dlg was certainly expressed in intraepithelial cervical, vulvar and laryngeal HPV‐associated lesions, its cellular and tissue distribution patterns were altered compared to normal tissue. However, marked reduction in Dlg levels was observed in HPV‐positive invasive cervical carcinomas. To elucidate whether the loss of Dlg was significant for carcinogenesis in general, we investigated Dlg expression in tumors not associated with HPV. In colon and kidney carcinomas, Dlg was expressed, albeit with a different pattern of distribution with respect to the normal tissue. The loss of Dlg may be considered a late‐stage marker in cervical carcinogenesis, but alterations in its expression and localization take place during the different dysplastic stages. Dlg downregulation and/or alterations in its localization may contribute to transformation and may explain some of the characteristics of the malignant cells, such as loss of polarity and high migration ability.

New generic primer system targeting mucosal/genital and cutaneous human papillomaviruses leads to the characterization of HPV 115, a novel Beta-papillomavirus species 3

We explored the cutaneotropic HPV genetic diversity in 71 subjects from Argentina. New generic primers (CUT) targeting 88 mucosal/cutaneous HPV were designed and compared to FAP primers. Overall, 69 different HPV types/putative types were identified, being 17 of them novel putative types. Phylogenetic analysis of partial L1 sequences grouped 2 novel putative types in the Beta-PV, 14 in the Gamma-PV and 1 in the Mu-PV genera. CUT primers showed broader capacity than FAP primers in detecting different genera/species and novel putative types (p<0.01). Using overlapping PCR, the full-length genome of a Beta-PV putative type was amplified and cloned. The new virus, designated HPV 115, encodes five early genes and two late genes. Phylogenetic analysis indicated HPV 115 as the most divergent type within the genus Beta-PV species 3. This report is the first providing data on cutaneous HPVs circulating in South America and expands our knowledge of the Papillomaviridae family.

Analysis of the genetic diversity and phylogenetic relationships of putative human papillomavirus types.

More than 170 human papillomavirus (HPV) types have been completely sequenced, curated and divided into five genera: Alphapapillomavirus, Betapapillomavirus, Gammapapillomavirus, Mupapillomavirus and Nupapillomavirus. With the application of PCR methods, hundreds of putative novel HPV types have been identified as PCR amplicons in mucosa and skin. However, at present there are no studies reporting a systematic search of the currently known L1 amplicons and their phylogenetic relationships. This survey revealed the existence of at least 202 different putative HPV types that are pending for full-genome characterization: five alphapapillomaviruses, 37 betapapillomaviruses, 159 gammapapillomaviruses and one mupapillomavirus. All potential viruses of the genera Alphapapillomavirus and Betapapillomavirus were grouped in the defined species, while 59 putative gammapapillomaviruses types were segregated in 21 unidentified putative species. These data highlight the need for progress in the identification of additional taxa of the family Papillomaviridae in order to elucidate the diversity, evolution and medical implications of these viruses.

Identification of human papillomavirus type 156, the prototype of a new human gammapapillomavirus species, by a generic and highly sensitive PCR strategy for long DNA fragments.

This study developed a hanging-droplet long PCR, a generic and highly sensitive strategy to facilitate the identification of new human papillomavirus (HPV) genomes. This novel procedure used for the first time the hanging-droplet PCR technique for the amplification of long DNA fragments with generic primers targeting the L1 and E1 regions. It was first applied to the amplification of types belonging to the highly divergent genus Gammapapillovirus (γ-PV). The hanging-droplet long PCR was 100-fold more sensitive than a simple long PCR procedure, detecting as few as ten copies of HPV-4. Nineteen skin samples, potentially containing putative HPV types from the γ-PV genus, were also screened. The method identified four γ-PV genomic halves from new and previously described putative types, and made the full characterization of HPV-156 possible. This novel virus meets the criteria for a new species within the γ-PV genus, with nucleotide identities in the L1 ORF ranging from 58.3 to 67.3 % compared with representative types of the current γ-PV species. HPV-156 showed the highest identity to HPV-60 (67.3 %) from species γ-4, and was consistently closely related to it in both late- and early-gene-derived phylogenies. In conclusion, this report provides a versatile and highly sensitive approach that allowed identification of the prototype of a new species within the γ-PV genus. Its application with primers targeting the different genera in which both human and non-human PVs are distributed may facilitate characterization of the missing members of the family Papillomaviridae.

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.

Prevalence of human papillomavirus infection in Argentinean women attending two different hospitals prior to the implementation of the National Vaccination Program

Cervarix vaccine was included in the National Immunization Program of Argentina in 2011 but data about the local distribution of human papillomavirus (HPV) infection in women exposed to the virus are scarce. This cross‐sectional study determined the prevalence and type distribution of HPV infection in unvaccinated women attending routine gynecological screening in two public hospitals located in Buenos Aires and Santa Fe, Argentina. Socio‐demographic, sexual behavior, and co‐factors information was obtained from all participants (Buenos Aires, n = 429; Santa Fe, n = 433). Cervicovaginal swabs were tested with an MY11/09 primer‐based assay and with the CUT primer system targeting mucosal/cutaneous HPVs. Participants from Buenos Aires showed significantly higher rates of HPV infection (52.4% vs. 40.6%), of multiple infections (24.2% vs. 16.4%), and of low‐risk (20.3% vs. 13.9%) and high‐risk types (44.1% vs. 33.3%) than those from Santa Fe. HPV‐66 (Buenos Aires: 17%) and HPV‐16 (Santa Fe: 8.5%) were the most prevalent types. Novel HPV‐66 putative subtype and variants were identified. Vaccine types 16 and 18 were frequent (Buenos Aires: 13.5%; Santa Fe: 10.2%) but few participants had co‐infections with both (Buenos Aires: 1.4%; Santa Fe: 0.2%). A common risk factor for HPV infection was having a new sexual partner in the last year (Buenos Aires: OR 2.53, P < 0.001; Santa Fe: OR 1.85, P = 0.04). This study provides valuable baseline data for future assessment of the impact of massive vaccination in Argentina and it underlines the use of additional HPV testing strategies, such as the CUT system, for surveillance and vaccinology. J. Med. Virol. 85:655–666, 2013.

Corrigendum: 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. β-PVs (β-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 β-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 β-2. This provides useful data on cutaneous HPV infections in high UV-exposed regions.

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.

Molecular characterization, tissue tropism, and genetic variability of the novel Mupapillomavirus type HPV204 and phylogenetically related types HPV1 and HPV63.

HPV204 is the only newly identified Mupapillomavirus (Mu-PV) type in more than a decade. To comprehensively characterize HPV204, we performed a detailed molecular analysis of the viral genome and evaluated its clinical relevance in comparison to the other Mu-PVs, HPV1 and HPV63. The 7,227-bp long genome of HPV204 exhibits typical genomic organization of Mu-PVs with eight open reading frames (ORFs) (E6, E7, E1, E2, E8, E4, L2, and L1). We developed three type-specific quantitative real-time PCRs and used them to test a representative collection (n = 1,006) of various HPV-associated benign and malignant neoplasms, as well as samples of clinically normal cutaneous, mucosal, and mucocutaneous origins. HPV204, HPV1, and HPV63 were detected in 1.1%, 2.7%, and 1.9% of samples tested, respectively, and were present in skin and mucosa, suggesting dual tissue tropism of all Mu-PVs. To evaluate the etiological role of Mu-PVs in the development of HPV-associated neoplasms, Mu-PV viral loads per single cell were estimated. HPV1 and HPV63 were present in high viral copy numbers in 3/43 and 1/43 cutaneous warts, respectively, and were identified as the most likely causative agents of these warts. HPV204 viral load was extremely low in a single HPV204-positive cutaneous wart (7.4 × 10−7 viral copies/cell). Hence, etiological association between HPV204 and the development of cutaneous warts could not be established. To the best of our knowledge, this is the first study to evaluate the genetic variability of Mu-PVs by sequencing complete LCR genomic regions of HPV204, HPV1, and HPV63. We detected several nucleotide substitutions and deletions within the LCR genomic regions of Mu-PVs and identified two genetic variants of HPV204 and HPV63 and five genetic variants of HPV1.

Identificación de papilomavirus humanos en lesiones cutáneas benignas y malignas no melanoma por métodos moleculares

Los virus papiloma humano (PVH) estan presentes en la piel como flora normal, donde permanecen en forma latente, pudiendo desarrollar en ciertas oportunidades, lesiones cutaneas. Objetivo: Identificar los tipos de PVH con tropismo por epitelios cutaneos y analizar su posible asociacion con lesiones cutaneas benignas y carcinomas cutaneos no melanoma. Materiales y metodos: Estudio prospectivo realizado en el servicio de dermatologia del Hospital provincial del Centenario, desde Junio de 2007 a Noviembre de 2008. Se obtuvieron muestras mediante hisopados de regiones: fotoexpuesta (FE), no fotoexpuesta (NFE), perilesional (PL), superficie de lesion (SL) y biopsias de lesiones para estudio histopatologico. Se incluyeron pacientes derivados para estudio histopatologico de las lesiones antes referidas. Deteccion de PVH mediante PCR. Resultados: Participaron 67 pacientes, 41 hombres y 26 mujeres, edad promedio de 51 anos (rango: 19-89 anos). 300 muestras resultaron idoneas para la amplificacion por PCR. La frecuencia de ADN de PVH hallado fue del 58% (176/300) (Figura 1), encontrandose 75% en FE, 39% en NFE, 75% en PL, 66% en SL y 35% en las biopsias. Se identificaron 69 tipos diferentes de HPV, siendo mas frecuentes el 2, 21, 20 y 6. Se detecto un nuevo PVH, el 115. No se identifico PVH en muestras de carcinomas. En queratosis seborreicas se detecto en un 27%. Conclusiones: Se obtuvieron datos acerca de los PVH circulantes en los pacientes de nuestra region. Corroboramos la influencia de la radiacion ultravioleta sobre la infeccion por este virus, asi como su presencia en queratosis seborreicas. Identificamos un nuevo HPV en Sudamerica.