Human papillomaviruses (HPV) are etiologically linked with the development of various benign and malignant neoplasms in humans. As of November 2017, a total of 206 HPV types were officially recognized by the HPV Reference center at the Karolinska Institute in Sweden, although it has recently been shown that there are still at least 400 novel HPV types that have not yet been identified. In 2014, our research group identified a novel HPV, HPV-204, in an anal canal swab sample. HPV-204 was preliminarily classified into the genus Mupapillomavirus (Mu-PV) that consisted of only two HPV types: HPV-1 and HPV-63. In this study, our aim was to perform comprehensive phylogenetic and molecular characterization of HPV-204. In the second part of the study, we wanted to determine the tissue tropism and potential clinical significance of Mu-PVs by testing a representative collection of clinical specimens. Finally, we wanted to evaluate the genetic variability of Mu-PVs by amplifying and sequencing the long control region (LCR) of each Mu-PV.
Using several programs and freely available online applications, we determined characteristic genomic regions of HPV-204. The 7,227 base pair (bp) long genome of HPV-204 with a GC ratio of 37.8% exhibits typical genomic organization of Mu-PVs with eight open reading frames (ORFs) (E6, E7, E1, E2, E8, E4, L2, and L1), while lacking the E5 ORF. Based on phylogenetic analysis of the L1 gene of all officially recognized HPV types, HPV-204 clusters within the Mu-PV genus, species Mu-3. HPV-204 is most similar to HPV-63, since the L1 gene of HPV-204 exhibits 66.9% nucleotide sequence identity with the L1 gene of HPV-63 and 66.1% with the L1 gene of HPV-1.
To evaluate the tissue tropism and potential clinical relevance of Mu-PVs, we tested a representative collection of clinical samples (n=1,006) using three newly developed and highly sensitive quantitative type-specific real-time polymerase chain reactions (RT-PCRs) for detection of HPV-204, HPV-1, and HPV-63. A total of 11, 27, and 19 HPV-204, HPV-1, and HPV-63 isolates were obtained, respectively. HPV204 was detected in cervical swabs (1/116; 0.8%), cutaneous wart (1/43; 2.3%), swabs of the anal canal (4/110; 3.6%), and swabs of the penile surface (5/110; 4.5%). HPV-1 was detected in nasopharyngeal swabs (2/110; 1.8%), eyebrow hair follicle (1/110; 0.9%), cutaneous warts (21/43; 48.8%), and anogenital warts (3/40; 7.5%), while HPV-63 was detected in nasopharyngeal swabs (3/110; 2.7%), swabs of the oral cavity (2/105; 1.9%), eyebrow hair follicles (4/110; 3.6%), cutaneous warts (9/43; 20.9%), and swab of the anal canal (1/110; 0.9%). We have confirmed that all Mu-PVs exhibit dual tissue tropism, since HPV-204, HPV-1, and HPV-63 were detected in cutaneous, mucosal, and mucocutaneous epithelia.
HPV-204 viral load was extremely low in a single HPV-204-positive cutaneous wart (7.4 × 10−7 viral copies/cell). Hence, etiological association between infection with HPV-204 and the development of cutaneous warts could not be confirmed. Although the majority of HPV-1-positive samples contained low number of viral copies (range 5.9 x 10-6 – 1.8 x 10-2 viral copies/cell), HPV-1 was the most likely causative agent of three cutaneous warts with high HPV-1 viral load (range 8.5 x 103 – 5.2 x 104 viral copies/cell). Similarly, HPV-63 viral load was low in the majority of HPV-63-positive tissue samples (range 3.2 x 10-6 – 1.1 x 10-2 viral copies/cell). However, HPV-63 was the most likely causative agent of one common wart, where it was present in high viral copy numbers (3.9 x 102 viral copies/cell). Thus, we have confirmed that the majority of infections with Mu-PVs are clinically latent, whereas HPV-1 and HPV-63 may be the causative agents of sporadic cases of common warts. In contrast, we were not able to establish a causative association between the presence of HPV-204 and the development of common warts.
Finally, we evaluated the genetic variability of Mu-PVs by sequencing complete LCR genomic regions of HPV-204, HPV-1, and HPV-63. We identified one genetic variant of HPV-204 that contained a single nucleotide polymorphism (SNP), six genetic variants of HPV-1 that contained 1-19 SNPs and/or nucleotide deletions and two genetic variants of HPV-63 that contained 6-13 SNPs and/or nucleotide deletions. Based on the obtained data, none of the genetic variants could be reliably associated with specific clinical manifestations or anatomical location of infection.
Part of the doctoral thesis was published as a scientific paper in a journal cited by the Science Citation Index (SCI) (IF2016=2.806):
Šterbenc A, Hošnjak L, Chouhy D, Bolatti EM, Oštrbenk A, Seme K, Kocjan BJ, Luzar B, Giri AA, Poljak M. Molecular characterization, tissue tropism, and genetic variability of the novel Mupapillomavirus type HPV204 and phylogenetically related types HPV1 and HPV63. PLoS One 2017;12:e0175892.