Identification of a Novel Human Papillomavirus, Type HPV199, Isolated from a Nasopharynx and Anal Canal, and Complete Genomic Characterization of Papillomavirus Species Gamma-12

Abstract

The novel human papillomavirus type 199 (HPV199) was initially identified in a nasopharyngeal swab sample obtained from a 25 year-old immunocompetent male. The complete genome of HPV199 is 7,184 bp in length with a GC content of 36.5%. Comparative genomic characterization of HPV199 and its closest relatives showed the classical genomic organization of Gammapapillomaviruses (Gamma-PVs). HPV199 has seven major open reading frames (ORFs), encoding five early (E1, E2, E4, E6, and E7) and two late (L1 and L2) proteins, while lacking the E5 ORF. The long control region (LCR) of 513 bp is located between the L1 and E6 ORFs. Phylogenetic analysis additionally confirmed that HPV-199 clusters into the Gamma-PV genus, species Gamma-12, additionally containing HPV127, HV132, HPV148, HPV165, and three putative HPV types: KC5, CG2 and CG3. HPV199 is most closely related to HPV127 (nucleotide identity 77%). The complete viral genome sequence of additional HPV199 isolate was determined from anal canal swab sample. Two HPV199 complete viral sequences exhibit 99.4% nucleotide identity. To the best of our knowledge, this is the first member of Gamma-PV with complete nucleotide sequences determined from two independent clinical samples. To evaluate the tissue tropism of the novel HPV type, 916 clinical samples were tested using HPV199 type-specific real-time PCR: HPV199 was detected in 2/76 tissue samples of histologically confirmed common warts, 2/108 samples of eyebrow hair follicles, 2/137 anal canal swabs obtained from individuals with clinically evident anal pathology, 4/184 nasopharyngeal swabs and 3/411 cervical swabs obtained from women with normal cervical cytology. Although HPV199 was found in 1.4% of cutaneous and mucosal samples only, it exhibits dual tissue tropism. According to the results of our study and literature data, dual tropism of all Gamma-12 members is highly possible.

Fig 1. Genomic organization of HPV199 showing genomic positions of viral genes E6, E7, E1, E2, E4, L1, L2 and the non-coding long control region (LCR) located between L1 and E6.

Fig 2. Localization of zinc-finger domains and binding domain for conserved retinoblastoma tumor suppressor protein (pRb) in E6 and E7 ORF of HPV199 and phylogenetically related HPV types.

The E6 and E7 ORFs of all HPV types belonging to Gamma-PV species 12 contain two and one zinc-finger domains (CxxC(x)29CxxC), respectively. The pRB binding motif (LxCxE) was identified only in the E7 ORF of HPV165, CG2, HPV148 and HPV132. Locations of zinc-finger domains are marked as boxes with oblique lines and locations of pRb binding domains as boxes with horizontal lines.

Fig 3. Phylogenetic analysis of HPV199.

The complete HPV genome sequences of 75 officially recognized HPV types belonging to the genus Gamma-PV and all putative Gamma-PV types were obtained and aligned and a maximum likelihood phylogenetic tree was constructed. The nucleotide sequences of two Beta-PVs, HPV5 and HPV8, were used to root the tree. The numbers in each branch are bootstrap support values and are given as percentages. Species Gamma-12 is indicated by a black box.