Human papillomavirus type 6 and 11 genetic variants found in 71 oral and anogenital epithelial samples from Australia

Abstract

Genetic variation of 49 human papillomavirus (HPV) 6 and 22 HPV11 isolates from recurrent respiratory papillomatosis (RRP) (n = 17), genital warts (n = 43), anal cancer (n = 6) and cervical neoplasia cells (n = 5), was determined by sequencing the long control region (LCR) and the E6 and E7 genes. Comparative analysis of genetic variability was examined to determine whether different disease states resulting from HPV6 or HPV11 infection cluster into distinct variant groups. Sequence variation analysis of HPV6 revealed that isolates cluster into variants within previously described HPV6 lineages, with the majority (65%) clustering to HPV6 sublineage B1 across the three genomic regions examined. Overall 72 HPV6 and 25 HPV11 single nucleotide variations, insertions and deletions were observed within samples examined. In addition, missense alterations were observed in the E6/E7 genes for 6 HPV6 and 5 HPV11 variants. No nucleotide variations were identified in any isolates at the four E2 binding sites for HPV6 or HPV11, nor were any isolates found to be identical to the HPV6 lineage A or HPV11 sublineage A1 reference genomes. Overall, a high degree of sequence conservation was observed between isolates across each of the regions investigated for both HPV6 and HPV11. Genetic variants identified a slight association with HPV6 and anogenital lesions (p = 0.04). This study provides important information on the genetic diversity of circulating HPV 6 and HPV11 variants within the Australian population and supports the observation that the majority of HPV6 isolates cluster to the HPV6 sublineage B1 with anogenital lesions demonstrating an association with this sublineage (p = 0.02). Comparative analysis of Australian isolates for both HPV6 and HPV11 to those from other geographical regions based on the LCR revealed a high degree of sequence similarity throughout the world, confirming previous observations that there are no geographically specific variants for these HPV types.

Figure 1. HPV6 LCR phylogenetic tree from Australia and other geographical regions.

A maximum likelihood (ML) tree was inferred from molecular phylogenetic analysis of 105 aligned LCR sequences using MEGA5 based on the Tamura-Nei model with 500 bootstrap replicates . These 105 sequences were derived from Australia (KC300093–KC300140, KC333888), Slovenia (FM876166–FM876210) and South Africa (JN573163–JN573174). Reference sequences are shown in grey for lineage A (X00203), and sublineages B1 (AF092932) and B3 (L41216). The 38 variants are named by the lineage and sublineage they cluster to. Lineage A was comprised of one Australian (A-2), one Slovenian (A-3) and two geographically shared (A-1, A-4) variants. Lineage B, specifically sublineage B1 was comprised of Australian (B1–4, B1–5, B1–9, B1–10, B1–12, B1–13, B1–14, B1–15, B1–16, B1–17, B1–18, B1–20, B1–21, B1–24, B1–25, B1–26, B1–27, B1–28, B1–29), Slovenian (B1–1, B1–3, B1–6, B1–7, B1–11, B1–19, B1–22) and geographically shared (B1–2, B1–8, B1–23 ) variants, and lineage B3 was comprised of Australian (B3–4, B3–5), South African (B3–1, B3–3) and geographically shared (B3–2). In cases where sequences were identical only one sequence from each counrty was use to represent each variant. In six instances variants from different geographical regions were identical; A–1, A–4 and B1–2 (Australia and Slovenia), B3–2 (Slovenia and South Africa), B1–8 and B1–23 (Australia, Slovenia and South Africa).

Figure 2. HPV11 LCR phylogenetic tree from Australia and other geographical regions.

A maximum likelihood (ML) tree was inferred from molecular phylogenetic analysis of 94 aligned LCR sequences using MEGA5 based on the Tamura-Nei model with 500 bootstrap replicates . These 94 sequences were derived from Australia (KC329850–KC329871), Slovenia (FN870625–FN870687), China (EU918768), Hungary (HE574701, HE574705, FR872717) and Thailand (JQ773408–JQ773412). The reference sequence (M14119) from sublineage A1 is shown in grey. The 21 variants are named by the lineage they cluster to. Sublineage A1 was comprised of one Slovenian variant (A1–1) and one geographically shared variant (A1–2). Sublineage A2 was comprised of 6 Australian (A2–3, A2–6, A2–13, A2–14, A2–16, A2–17), Slovenian(A2–1, A2–7, A2–10, A2–12, A2–15), Hungarian (A2–2), Thai(A2–9), and geographically shared (A2–4, A2–5, A2–8, A2–11, A2–18, A2–19) variants. In cases where sequences were identical only one sequence from that country was use to represent each variant. In seven instances variants from different geographical regions were also identical, again one sequence was used to represent each variant; A1–2 (China and Slovenia), A2–4 (Hungary and Thailand), A2–8 (Australia, Hungary and Slovenia), A2–5, A2–11, A2–18 (Australia and Slovenia), A2–19 (Australia, Slovenia and Thailand).