Intra-host sequence variability in human papillomavirus

Abstract

Human papillomaviruses (HPVs) co-evolve slowly with the human host and each HPV genotype displays epithelial tropisms. We assessed the evolution of intra HPV genotype variants within samples, and their association to anogenital site, cervical cytology and HIV status. Variability in the L1 gene of 35 HPV genotypes was characterized phylogenetically using maximum likelihood, and portrayed by phenotype. Up to a thousand unique variants were identified within individual samples. In-depth analyses of the most prevalent genotypes, HPV16, HPV18 and HPV52, revealed that the high diversity was dominated by a few abundant variants. This suggests high intra-host mutation rates. Clades of HPV16, HPV18 and HPV52 were associated to anatomical site and HIV co-infection. Particularly, we observed that one HPV16 clade was specific to vaginal cells and one HPV52 clade was specific to anal cells. One major HPV52 clade, present in several samples, was strongly associated with cervical neoplasia. Overall, our data suggest that tissue tropism and HIV immunosuppression are strong shapers of HPV evolution.

Keywords: Anogenital; HIV; HPV phylogenetics; HPV variability; Tissue tropism.

Graphical abstract

Fig. 1

Summary of phylogenetic tree construction from 144 women who each provided duplicate samples (anal + vaginal swabs). The L1 region of HPV was sequenced using next generation sequencing on the Illumina MiSeq platform. The sequence pairs generated were filtered, clustered and aligned to produce 35 phylogenetic trees. HPV 16, 18 and 52 were further analysed for in-depth statistical description of the distribution of clades by site of HPV infection, cervical cytology result and HIV statuses of the women.

Fig. 2

Distribution of sequences per variant. In HPV genotypes A) HPV16, B) HPV18 and c) HPV52 for all samples included in the trees. Each sample is represented by a unique colour.

Fig. 3

Percentage of the minor allele throughout the amplified region of L1 for HPV 16, 18 and 52. Each nucleotide is represented by a unique colour.

Fig. 4

HPV 16 phylogenetic tree was constructed from 38 samples from 34 women and a total of 4275 variants using maximum likelihood (RAxML). The best tree was created from four multiple runs and 10,000 boot-straps and presented in ‘Fan’ mode. Two clades (X and Y) are observed. The tree was colour based on four variables; A) sample, B) site (vaginal or anal), C) cervical cytology outcome and D) HIV status. Cytology outcome were encoded according to Bethesda classification; Negative = Negative = Negative for intra-epithelial lesions and malignancy (NILM), High grade = High = High grade squamous intra-epithelial lesions (HGSIL) and Atypical squamous cells, cannot exclude HGSIL (ASC-H), Low grade = Low‐grade = Low-grade squamous intraepithelial lesion (LGSIL) and Atypical squamous cells of undetermined significance (ASC-US) and Unsatisfactory = slide = slide was not satisfactory for evaluation.

Fig. 5

The HPV 18 phylogenetic tree was constructed from a total of 2403 variants of 41 samples from 37 women using maximum likelihood (RAxML). The best tree was created from four multiple runs and 10,000 bootstraps and presented in ‘Fan’ mode. Two clades (X and Y) are observed. The tree was color based on four variables; A) sample, B) site (vaginal or anal), C) cervical cytology outcome and D) HIV status. Cytology outcome were encoded according to Bethesda classification; Negative = Negative for intra-epithelial lesions and malignancy (NILM), High grade = High grade squamous intra-epithelial lesions (HGSIL) and Atypical squamous cells, cannot exclude HGSIL (ASC-H), Low grade = Low-grade squamous intraepithelial lesion (LGSIL) and Atypical squamous cells of undetermined significance (ASC-US) and Unsatisfactory = slide was not satisfactory for evaluation.

Fig. 6

The HPV 52 phylogenetic tree was constructed from a total of 4728 variants of 53 samples from 44 women using maximum likelihood (RAxML). The best tree was created from four multiple runs and 10,000 bootstraps and presented in ‘Fan’ mode. Three distinct clades were observed, Clades X, Y and Z. The tree was color based on four variables; A) sample, B) site (vaginal or anal), C) cervical cytology outcome and D) HIV status. Cytology outcome were encoded according to Bethesda classification; Negative = Negative for intra-epithelial lesions and malignancy (NILM), High grade = High-grade squamous intra-epithelial lesions (HGSIL) and Atypical squamous cells, cannot exclude HGSIL (ASC-H), Low grade = Low-grade squamous intraepithelial lesion (LGSIL) and Atypical squamous cells of undetermined significance (ASC-US) and Unsatisfactory = slide was not satisfactory for evaluation.