Global Genomic Diversity of Human Papillomavirus 6 Based on 724 Isolates and 190 Complete Genome Sequences

Abstract

Human papillomavirus type 6 (HPV6) is the major etiological agent of anogenital warts and laryngeal papillomas and has been included in both the quadrivalent and nonavalent prophylactic HPV vaccines. This study investigated the global genomic diversity of HPV6, using 724 isolates and 190 complete genomes from six continents, and the association of HPV6 genomic variants with geographical location, anatomical site of infection/disease, and gender. Initially, a 2,800-bp E5a-E5b-L1-LCR fragment was sequenced from 492/530 (92.8%) HPV6-positive samples collected for this study. Among them, 130 exhibited at least one single nucleotide polymorphism (SNP), indel, or amino acid change in the E5a-E5b-L1-LCR fragment and were sequenced in full. A global alignment and maximum likelihood tree of 190 complete HPV6 genomes (130 fully sequenced in this study and 60 obtained from sequence repositories) revealed two variant lineages, A and B, and five B sublineages: B1, B2, B3, B4, and B5. HPV6 (sub)lineage-specific SNPs and a 960-bp representative region for whole-genome-based phylogenetic clustering within the L2 open reading frame were identified. Multivariate logistic regression analysis revealed that lineage B predominated globally. Sublineage B3 was more common in Africa and North and South America, and lineage A was more common in Asia. Sublineages B1 and B3 were associated with anogenital infections, indicating a potential lesion-specific predilection of some HPV6 sublineages. Females had higher odds for infection with sublineage B3 than males. In conclusion, a global HPV6 phylogenetic analysis revealed the existence of two variant lineages and five sublineages, showing some degree of ethnogeographic, gender, and/or disease predilection in their distribution. IMPORTANCEThis study established the largest database of globally circulating HPV6 genomic variants and contributed a total of 130 new, complete HPV6 genome sequences to available sequence repositories. Two HPV6 variant lineages and five sublineages were identified and showed some degree of association with geographical location, anatomical site of infection/disease, and/or gender. We additionally identified several HPV6 lineage-and sublineage-specific SNPs to facilitate the identification of HPV6 variants and determined a representative region within the L2 gene that is suitable for HPV6 whole-genome-based phylogenetic analysis. This study complements and significantly expands the current knowledge of HPV6 genetic diversity and forms a comprehensive basis for future epidemiological, evolutionary, functional, pathogenicity, vaccination, and molecular assay development studies.

Global Genomic Diversity of Human Papillomavirus 11 Based on 433 Isolates and 78 Complete Genome Sequences

“…In accordance with our findings from an HPV6 global genomic diversity study (29), the topology of the HPV11 phylogenetic tree constructed from concatenated E5a-E5b-L1-URR sequences resembled the topology of the tree constructed from the complete HPV11 genome. On the other hand, the topologies of the trees constructed with each of the nine individual ORFs and the URR were dichotomous, clearly identifying lineages A and B but poorly resolving sublineages A1 to A4.…”

“…Due to the identification of a novel lineage B, originating from a single African isolate, the calculated maximum pairwise dissimilarly of the complete HPV11 genome variants (e.g., isolate LT4 versus isolate SA3408) reached 1.3%, exceeding the previously reported rate of diversity by approximately 4-fold (25,26,28). Hence, the genomic diversity of HPV11 of 1.3% is highly similar to that of its closest relative, HPV6 (1.6%) (29). It is distinctly lower than that of the two most important high-risk HPV types, HPV16 (2.3%) and HPV18 (2.1%), and similar to that of some of their closest relatives, HPV31 (1.4%), HPV45 (1.5%), and HPV70 (1.6%) (43)(44)(45).…”

“…As with HPV6, we strongly believe that the absence of a strong geographical clustering of HPV11 genomic variants is most probably due to the lower replication rate and delayed fixation of (sub)lineage-specific SNPs throughout the HPV11 genome (24,26,29,43,51). Therefore, the major HPV11 lineages and sublineages may have evolved well before humans spread to all the continents.…”

“…For the purpose of this study, a total of 207 HPV11 isolates were collected from 14 countries covering six continents, and four genomic regions, including the E5a, E5b, and L1 ORFs and the noncoding region URR were sequenced. These particular regions were selected for initial sequencing on the basis of findings from previous studies: (i) E5a, L1, and URR contain sequence information sufficient for unambiguous differentiation between HPV11 sublineages A1 and A2 (25,26,28); (ii) the E5a and E5b genes evolve faster and mutate more frequently than other HPV genes (40,41); and (iii) our recent genomic diversity study of HPV6 (the closest relative of HPV11) showed that the concatenated E5a-E5b-L1-URR sequence can be reliably used as a surrogate for the whole-genome phylogenetic clustering of HPV6 variants (29), and thus, sequencing of the same genomic regions would additionally allow comparison of the genetic diversity of a large number of HPV6 and HPV11 isolates. The genomes of a total of 30 HPV11 isolates showing the highest diversity in the E5a, E5b, L1, and/or URR genomic region were fully sequenced and phylogenetically evaluated together with 48 complete HPV11 genome sequences from GenBank and PaVE, of which 70.1% (34/48) mainly originated from two pre-FIG 1 HPV11 heat map and maximum likelihood trees inferred from 78 complete HPV11 genomes.…”

“…The final extension step was carried out at 68°C for 7 min. PCR products were verified by gel electrophoresis, purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany), and submitted to Macrogen Europe (Amsterdam, Netherlands) or to the in-house sequencing facility of the Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, for Sanger sequencing of both strands using previously published primers (25) and a previously described sequencing protocol (29).…”