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

Abstract

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 (c-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 c-PV genus, were also screened. The method identified four c-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 c-PV genus, with nucleotide identities in the L1 ORF ranging from 58.3 to 67.3 % compared with representative types of the current c-PV species. HPV-156 showed the highest identity to HPV-60 (67.3 %) from species c-4, and was consistently closely related to it in both late-and earlygene-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 c-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.

Identification and Characterization of Eleven Novel Human Gamma-Papillomavirus Isolates from Healthy Skin, Found at Low Frequency in a Normal Population

“…The ATP binding site is modified slightly in HPV 164 and HPV 167 as “GPPD S GKS”, in HPV 169 as “G V PDSGKS”, and in HPV 170 as “GPP N TGKS” (the amino acid in bold indicate mutation site). The length of the upstream regulatory region (URR) is from 447 bp (HPV 165) to 581 bp (HPV 170) (Table 1), similar to the size of URRs in other HPV types of the genus Gamma-papillomavirus [9,12]. Within the URR region of all these novel HPV types, putative E2-binding sites (ACCN 6 GGT) were identified (Table S1).…”

“…Isolation and characterization novel HPV genomes will reveal the existence of distinct human papillomavirus (HPV) types and will provide basic information for understanding the biologic significance and the basis of HPV evolution. Many studies focus on identifying novel HPV from common warts, keratotic lesions or cutaneous squamous cell carcinoma [9-14], but few studies have investigated novel HPV types originated from healthy individuals. …”

Complete Genome Sequence of Phodopus sungorus Papillomavirus Type 1 (PsPV1), a Novel Member of the Pipapillomavirus Genus, Isolated from a Siberian Hamster

“…Partial PsPV1 L1 and E1 gene sequences were initially obtained from the lesion using the hanging-droplet PCR technique with FAP64/FAP6085F (2) and E1Gamma-F/E1Gamma-R primers (3), respectively. A BLAST comparison (http://blast.ncbi.nlm.nih.gov/Blast.cgi) of the partial L1 sequence revealed that PsPV1 clusters to the genus Pipapillomavirus .…”

Novel Human Gammapapillomavirus Species in a Nasal Swab

“…The L1 gene of HPV-Fin864 showed a best BLASTn match to HPV-156, a recently described ␥-HPV species found in skin samples from immunocompetent patients (11). The Gammapapillomavirus genus currently consists of 10 viral species known to infect humans (1).…”

“…The Gammapapillomavirus genus currently consists of 10 viral species known to infect humans (1). Recently published genomes are expected to increase that number (11,13). According to the International Committee on Taxonomy of Viruses (ICTV), the members of a papillomavirus species should share at least 70% nucleotide identity in the L1 gene (12).…”