P01-01HPV16 MINORITY VARIANTS AMONG CERVICAL AND ANAL SAMPLES WITH SINGLE HPV16 OR MULTIPLE HPV TYPES INFECTIONS

01. Viral and molecular biology
A.A. Mariaggi 1, H. Péré 2, B. Visseaux 1, D. Veyer 2, V. Joly 3, Q. Le Hingrat 1, A. Couvelard 4, M. Bucau 4, C. Davitian 5, L. Abramowitz 6, D. Descamps 1, C. Charpentier 1.
1IAME, UMR 1137, INSERM, Université Paris Diderot, Sorbonne Paris Cité, AP-HP, Laboratoire de Virologie, Hôpital Bichat, AP-HP, Paris, France (France), 2Laboratoire de Virologie, Université Paris Descartes, Hôpital Européen Georges Pompidou, AP-HP, Paris, France (France), 3IAME, UMR 1137, INSERM, Université Paris Diderot, Sorbonne Paris Cité, AP-HP, Service de Maladies Infectieuses et Tropicales, Hôpital Bichat, AP-HP, Paris, France (France), 4Université Paris Diderot, Sorbonne Paris Cité, AP-HP, Laboratoire d'Anatomo-CytoPathologie, Hôpital Bichat, AP-HP, Paris, France (France), 5Service de Gynécologie-Obstétrique, Hôpital Bichat, AP-HP, Paris, France (France), 6Service d'Hépato-Gastro-Entérologie, Hôpital Bichat, AP-HP, Paris, France (France)

Background / Objectives

To assess presence of minority variants (MV) in HPV16 genomes isolated from clinical cervical and anal specimens of patients followed in Paris area, France, using ultra-deep sequencing of the whole viral genome.


Methods

HPV detection and typing was performed with Anyplex®II HPV28 detection kit (Seegene). Ultra-deep sequencing was performed using Illumina® platform. HPV16 lineages were determined by phylogenetic analysis using RAxML. HPV16 viral loads were determined by “in house” real-time PCR assays. Viral genome integration ratio was assessed by E2/E6 ratio.


Results

We assessed 44 consecutive smears routine samples (28 cervical and 16 anal [n=12 men]) positive for HPV16. 19 patients (43%) were HIV-infected. Cytohistologic data showed 10 LSIL and 5 HSIL in cervical samples, 3 LSIL and 8 HSIL in anal samples. Overall, multiple HPV (mHPV) infections (high risk [hr] and/or low risk [lr]) were observed in 32 samples (73%). In anal samples mHPV infections were present in a higher proportion (94% vs 61%, p=0.03) and with a higher diversity of hrHPV types (p=0.04) than in cervical samples. Phylogenetic analysis showed diversity in HPV16 lineage variants with 32 A lineage (73%), 3 B (7%), 7 C (16%) and 2 D (4%). Ultra-deep sequencing analysis revealed that nucleotidic non synonymous substitutions were present in minority proportion (i.e. ranging from 2 to 20%) in 14 samples (32%), without difference between cervical and anal samples (32% and 31%, respectively). Viral MV were present at the median proportion of 3.4% (IQR=2.4-6.4). Most of MV (64%) presented only a single nucleotide variation on the whole genome, located in E1 or E2 regions in most of cases (86%). Among cervical specimens, a lower frequency of lrHPV coinfections was observed in samples displaying MV than in samples without MV (11% vs 58%, p=0.04). A lower frequency of hrHPV coinfections was also observed but was no significant (33% vs 63%). In addition, patients with mHPV cervical infections had more frequently high HPV16 viral load (i.e. >50 copies/cell) than patients without mHPV infections (77% vs 20%, p=0.012). The proportion of patients with a high integrated viral genome ratio (i.e. >50%) increased with the cervical lesion grade: 44%, 56% and 80% in ASC-US, LSIL and HSIL, respectively. Regarding anal samples, all except one had mHPV infections, 50% were HSIL and 55% had a high integrated viral genome ratio.


Conclusion

This first large study of HPV16 whole genome ultra-deep sequencing showed presence of viral MV in one third of the samples, in cervical as well as in anal specimens. In addition, we evidenced that, in almost all cases, cervical samples with MV had no lrHPV coinfections.


References