Ramón Fernández Bussy

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.

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.

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.

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.