UNLABELLED: 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. IMPORTANCE: This 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.

Department of Experimental Virology National Reference Laboratory for Papillomaviruses Institute of Hematology and Blood Transfusion Prague Czech Republic

Department of Laboratory Medicine Lithuanian University of Health Sciences Medical Academy Kaunas Lithuania

Department of Medical Microbiology and Virology Faculty of Health Sciences University of the Free State Bloemfontein South Africa

Department of Microbiology and Infectious Diseases The Royal Women's Hospital Parkville Victoria Australia Department of Obstetrics and Gynaecology University of Melbourne Victoria Australia Murdoch Children's Research Institute Parkville Victoria Australia

Department of Microbiology Faculty of Medicine Chinese University of Hong Kong Prince of Wales Hospital Hong Kong Special Administrative Region China

Department of Molecular Diagnostics University Hospital for Infectious Diseases Dr Fran Mihaljević Zagreb Croatia

Department of Obstetrics and Gynaecology Cardiff University School of Medicine Institute of Cancer and Genetics Cardiff United Kingdom

Department of Oncology Division of Cancer Epidemiology McGill University Montréal Québec Canada

Department of Otolaryngology Lithuanian University of Health Sciences Medical Academy Kaunas Lithuania

Department of Otorhinolaryngology Faculty of Health Sciences University of the Free State Bloemfontein South Africa

Department of Pathology McGill University and Jewish General Hospital Montréal Québec Canada

Department of Pediatrics Albert Einstein College of Medicine New York New York USA

Departments of Microbiology and Immunology Epidemiology and Population Health Obstetrics and Gynecology and Women's Health Albert Einstein College of Medicine New York New York USA Department of Pediatrics Albert Einstein College of Medicine New York New York USA

Dermatology Research Centre The Chinese University of Hong Kong Prince of Wales Hospital Hong Kong Special Administrative Region China Department of Health Social Hygiene Service Centre for Health Protection Hong Kong Special Administrative Region China

DNA Laboratories Sdn Bhd UKM MTDC Technology Centre Universti Kebangsaan Malaysia Bangi Malaysia

Ganshintetsu Memorial Laboratory Department of Virology 2 National Institute of Health Tokyo Japan

Histocompatibility and Molecular Genetics Laboratory Dr Julio C Perrando Hospital Resistencia Chaco Argentina

Human Virology Group National Council of Scientific and Technical Research Institute of Molecular and Cell Biology of Rosario Rosario Argentina

Institute of Microbiology and Immunology Faculty of Medicine University of Ljubljana Ljubljana Slovenia

Laboratoire de Virologie Moléculaire Centre de Recherche Centre Hospitalier de L'Université de Montréal Hôpital Notre Dame Pavillon Deschamps Montréal Québec Canada

Lausanne University Hospital Institute of Microbiology Lausanne Switzerland

Medizinisches Versorgungszentrum wagnerstibbe für Laboratoriumsmedizin und Pathologie GmbH Hannover Germany

Oncogenic Viruses Service National Institute of Infectious Diseases ANLIS Dr Carlos G Malbrán Buenos Aires Argentina

Queen's Cancer Research Institute Queen's University Kingston Ontario Canada

Viral Exanthemata and STD Section National Microbiology Laboratory Public Health Agency of Canada Winnipeg Manitoba Canada

See more in PubMed

Bernard HU, Burk RD, Chen Z, van Doorslaer K, zur Hausen H, de Villiers EM. 2010. Classification of papillomaviruses (PVs) based on 189 PV types and proposal of taxonomic amendments. Virology 401:70–79. 10.1016/j.virol.2010.02.002 PubMed DOI PMC

Aubin F, Prétet JL, Jacquard AC, Saunier M, Carcopino X, Jaroud F, Pradat P, Soubeyrand B, Leocmach Y, Mougin C, Riethmuller D. 2008. Human papillomavirus genotype distribution in external acuminata condylomata: a large French national study (EDiTH IV). Clin. Infect. Dis. 47:610–615. 10.1086/590560 PubMed DOI

Bouvard V, Baan R, Straif K, Grosse Y, Secretan B, El Ghissassi F, Benbrahim-Tallaa L, Guha N, Freeman C, Galichet L, Cogliano V. 2009. A review of human carcinogens. Part B: biological agents. Lancet Oncol. 10:321–322. 10.1016/S1470-2045(09)70096-8 PubMed DOI

Garland SM, Steben M, Sings HL, James M, Lu S, Railkar R, Barr E, Haupt RM, Joura EA. 2009. Natural history of genital warts: analysis of the placebo arm of 2 randomized phase III trials of a quadrivalent human papillomavirus (types 6, 11, 16, and 18) vaccine. J. Infect. Dis. 199:805–814. 10.1086/597071 PubMed DOI

Komloš KF, Kocjan BJ, Košorok P, Luzar B, Meglič L, Potočnik M, Hočevar-Boltežar I, Gale N, Seme K, Poljak M. 2012. Tumor-specific and gender-specific pre-vaccination distribution of human papillomavirus types 6 and 11 in anogenital warts and laryngeal papillomas: a study on 574 tissue specimens. J. Med. Virol. 84:1233–1241. 10.1002/jmv.23318 PubMed DOI

Cubie HA. 2013. Diseases associated with human papillomavirus infection. Virology 445:21–34. 10.1016/j.virol.2013.06.007 PubMed DOI

Cornall AM, Roberts JM, Garland SM, Hillman RJ, Grulich AE, Tabrizi SN. 2013. Anal and perianal squamous carcinomas and high-grade intraepithelial lesions exclusively associated with “low-risk” HPV genotypes 6 and 11. Int. J. Cancer 133:2253–2258. 10.1002/ijc.28228 PubMed DOI

Insinga RP, Liaw KL, Johnson LG, Madeleine MM. 2008. A systematic review of the prevalence and attribution of human papillomavirus types among cervical, vaginal, and vulvar precancers and cancers in the United States. Cancer Epidemiol. Biomarkers Prev. 17:1611–1622. 10.1158/1055-9965.EPI-07-2922 PubMed DOI PMC

Miralles-Guri C, Bruni L, Cubilla AL, Castellsague X, Bosch FX, de Sanjosé S. 2009. Human papillomavirus prevalence and type distribution in penile carcinoma. J. Clin. Pathol. 62:870–878. 10.1136/jcp.2008.063149 PubMed DOI

Huebbers CU, Preuss SF, Kolligs J, Vent J, Stenner M, Wieland U, Silling S, Drebber U, Speel EJ, Klussmann JP. 2013. Integration of HPV6 and downregulation of AKR1C3 expression mark malignant transformation in a patient with juvenile-onset laryngeal papillomatosis. PLoS One 8:e57207. 10.1371/journal.pone.0057207 PubMed DOI PMC

Bzhalava D, Guan P, Franceschi S, Dillner J, Clifford G. 2013. A systematic review of the prevalence of mucosal and cutaneous human papillomavirus types. Virology 445:224–231. 10.1016/j.virol.2013.07.015 PubMed DOI

Kiatpongsan S, Campos NG, Kim JJ. 2012. Potential benefits of second-generation human papillomavirus vaccines. PLoS One 7:e48426. 10.1371/journal.pone.0048426 PubMed DOI PMC

Combrinck CE, Seedat RY, Randall C, Roodt Y, Burt FJ. 2012. Novel HPV-6 variants of human papillomavirus causing recurrent respiratory papillomatosis in southern Africa. Epidemiol. Infect. 140:1095–1101. 10.1017/S0950268811001580 PubMed DOI

Danielewski JA, Garland SM, McCloskey J, Hillman RJ, Tabrizi SN. 2013. Human papillomavirus type 6 and 11 genetic variants found in 71 oral and anogenital epithelial samples from Australia. PLoS One 8:e63892. 10.1371/journal.pone.0063892 PubMed DOI PMC

de Matos RP, Sichero L, Mansur IM, do Bonfim CM, Bittar C, Nogueira RL, Küpper DS, Valera FC, Nogueira ML, Villa LL, Calmon MF, Rahal P. 2013. Nucleotide and phylogenetic analysis of human papillomavirus types 6 and 11 isolated from recurrent respiratory papillomatosis in Brazil. Infect. Genet. Evol. 16:282–289. 10.1016/j.meegid.2012.12.033 PubMed DOI

Heinzel PA, Chan SY, Ho L, O'Connor M, Balaram P, Campo MS, Fujinaga K, Kiviat N, Kuypers J, Pfister H. 1995. Variation of human papillomavirus type 6 (HPV-6) and HPV-11 genomes sampled throughout the world. J. Clin. Microbiol. 33:1746–1754 PubMed PMC

Kocjan BJ, Jelen MM, Maver PJ, Seme K, Poljak M. 2011. Pre-vaccination genomic diversity of human papillomavirus genotype 6 (HPV 6): a comparative analysis of 21 full-length genome sequences. Infect. Genet. Evol. 11:1805–1810. 10.1016/j.meegid.2011.06.022 PubMed DOI

Kocjan BJ, Poljak M, Cimerman M, Gale N, Potočnik M, Bogovac Ž, Seme K. 2009. Prevaccination genomic diversity of human papillomavirus genotype 6 (HPV 6). Virology 391:274–283. 10.1016/j.virol.2009.06.030 PubMed DOI

Burk RD, Chen Z, Harari A, Smith BC, Kocjan BJ, Maver PJ, Poljak M. 2011. Classification and nomenclature system for human Alphapapillomavirus variants: general features, nucleotide landmarks and assignment of HPV6 and HPV11 isolates to variant lineages. Acta Dermatovenerol. Alp. Panonica Adriat. 20:113–123 http://www.zsd.si/ACTA/PUBLIC_HTML/acta-apa-11-3/2.pdf PubMed PMC

Platt AR, Woodhall RW, George AL., Jr 2007. Improved DNA sequencing quality and efficiency using an optimized fast cycle sequencing protocol. Biotechniques 43:58, 60, 62. 10.2144/000112499 PubMed DOI

Kocjan BJ, Gale N, Hočevar Boltežar I, Seme K, Fujs Komloš K, Hošnjak L, Maver PJ, Jelen MM, Zupanič Pajnič I, Balažic J, Poljak M. 2013. Identical human papillomavirus (HPV) genomic variants persist in recurrent respiratory papillomatosis for up to 22 years. J. Infect. Dis. 207:583–587. 10.1093/infdis/jis733 PubMed DOI

Ure AE, Forslund O. 2012. Lack of methylation in the upstream region of human papillomavirus type 6 from aerodigestive tract papillomas. J. Virol. 86:13790–13794. 10.1128/JVI.01938-12 PubMed DOI PMC

Katoh K, Toh H. 2010. Parallelization of the MAFFT multiple sequence alignment program. Bioinformatics 26:1899–1900. 10.1093/bioinformatics/btq224 PubMed DOI PMC

Stamatakis A, Hoover P, Rougemont J. 2008. A rapid bootstrap algorithm for the RAxML Web servers. Syst. Biol. 57:758–771. 10.1080/10635150802429642 PubMed DOI

Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61:539–542. 10.1093/sysbio/sys029 PubMed DOI PMC

Drummond AJ, Rambaut A. 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 7:214. 10.1186/1471-2148-7-214 PubMed DOI PMC

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28:2731–2739. 10.1093/molbev/msr121 PubMed DOI PMC

Maddison DR, Maddison WP. 2005. MacClade 4: analysis of phylogeny and character evolution, version 4.08a. http://macclade.org/index.html PubMed

Smith B, Chen Z, Reimers L, van Doorslaer K, Schiffman M, Desalle R, Herrero R, Yu K, Wacholder S, Wang T, Burk RD. 2011. Sequence imputation of HPV16 genomes for genetic association studies. PLoS One 6:e21375. 10.1371/journal.pone.0021375 PubMed DOI PMC

Chen Z, Schiffman M, Herrero R, DeSalle R, Anastos K, Segondy M, Sahasrabuddhe VV, Gravitt PE, Hsing AW, Burk RD. 2013. Evolution and taxonomic classification of alphapapillomavirus 7 complete genomes: HPV18, HPV39, HPV45, HPV59, HPV68 and HPV70. PLoS One 8:e72565. 10.1371/journal.pone.0072565 PubMed DOI PMC

Chen Z, Schiffman M, Herrero R, Desalle R, Anastos K, Segondy M, Sahasrabuddhe VV, Gravitt PE, Hsing AW, Burk RD. 2011. Evolution and taxonomic classification of human papillomavirus 16 (HPV16)-related variant genomes: HPV31, HPV33, HPV35, HPV52, HPV58 and HPV67. PLoS One 6:e20183. 10.1371/journal.pone.0020183 PubMed DOI PMC

Ho L, Chan SY, Burk RD, Das BC, Fujinaga K, Icenogle JP, Kahn T, Kiviat N, Lancaster W, Mavromara-Nazos P. 1993. The genetic drift of human papillomavirus type 16 is a means of reconstructing prehistoric viral spread and the movement of ancient human populations. J. Virol. 67:6413–6423 PubMed PMC

Ong CK, Chan SY, Campo MS, Fujinaga K, Mavromara-Nazos P, Labropoulou V, Pfister H, Tay SK, ter Meulen J, Villa LL. 1993. Evolution of human papillomavirus type 18: an ancient phylogenetic root in Africa and intratype diversity reflect coevolution with human ethnic groups. J. Virol. 67:6424–6431 PubMed PMC

Chen Z, Terai M, Fu L, Herrero R, DeSalle R, Burk RD. 2005. Diversifying selection in human papillomavirus type 16 lineages based on complete genome analyses. J. Virol. 79:7014–7023. 10.1128/JVI.79.11.7014-7023.2005 PubMed DOI PMC

Sichero L, Ferreira S, Trottier H, Duarte-Franco E, Ferenczy A, Franco EL, Villa LL. 2007. High grade cervical lesions are caused preferentially by non-European variants of HPVs 16 and 18. Int. J. Cancer 120:1763–1768. 10.1002/ijc.22481 PubMed DOI

Bernard HU, Calleja-Macias IE, Dunn ST. 2006. Genome variation of human papillomavirus types: phylogenetic and medical implications. Int. J. Cancer 118:1071–1076. 10.1002/ijc.21655 PubMed DOI

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