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

CHOUHY, DIEGO; BOLATTI, ELISA; PICCIRILLI, GUSTAVO; SANCHEZ, ADRIANA; FERNANDEZ BUSSY, RAMÓN; GIRI ADRIANA A

JOURNAL OF GENERAL VIROLOGY

SOC GENERAL MICROBIOLOGY

Lugar: London; Año: 2013 vol. 94 p. 524 - 533

0022-1317

We developed the "Hanging droplet" long PCR, a generic and highly sensitive strategy to facilitate the identification of new human papillomavirus (HPV) genomes. The 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. We first applied it for the amplification of types belonging to the highly divergent γ-PV genus. The "Hanging droplet" long PCR was 100-fold more sensitive than the simple long PCR procedure, detecting down to 10 copies of HPV-4. We further screened 19 skin samples potentially containing putative HPV types from the γ-PV genus. The method identified 4 γ-PV genomic halves from new and previously described putative types, and made the full characterization of HPV-156 possible. The new virus meets the criteria for a new species within the γ-PV genus, showing nucleotide identities in the L1 ORF ranging from 58.3% to 67.3% 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 identifying 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 the characterization of the missing members of the family Papillomaviridae.