Complete Genomes of Human Papillomavirus Type 16 Viruses Isolated from Cases of Cervical Neoplasia and Squamous Cell Carcinomas Followed in Latvia in 2012-2024

Background: Persistent high-risk human papillomavirus (hrHPV) infection causes over 99% of cervical precancers and cancers worldwide, with HPV genotype 16 (HPV16) responsible for 50% of the cases. Latvia ranks among the top EU countries for cervical cancer incidence and mortality. In the general Latvian population, 4.2% women are hrHPV-infected, mostly with HPV16. However, information on the circulating HPV16 isolates is missing. Objectives: To study the genomic variability of the Latvian HPV16 isolates, compare them with HPV16 in Europe and across the globe, reveal features associated with the severity of cervical disease and uncover eventual sequence changes due the national HPV vaccination. Methods: DNA was extracted from the formalin-fixed paraffin-embedded cervical tissues of women diagnosed with cervical intraepithelial neoplasia (CIN) stages I-III and squamous cell carcinoma (SCC) grades 1-3 collected between 2012 and 2024. Samples positive for HPV16 were subjected to whole genome sequencing (WGS) on the Illumina platform (n=16) or Sanger sequencing of the E6/E7 coding region (n=31). Consensus HPV16 sequence was generated, and single nucleotide polymorphisms (SNP) and eventual amino acid substitutions (AAS) were analysed. Results: Complete genomes of 16 HPV16 variants were reconstructed, 13 related to the European sublineage A1, and 3, to the sublineage A2 references. Sequences showed high conservation, still 93 non-redundant variants were identified. The highest variability was observed for the capsid protein L2, the lowest, for oncoprotein E7. Prevalence of SNPs and AAS in the Latvian HPV16 variants, specifically in capsid protein L1, did not increase with time, showing no effect of HPV vaccination. Associations between HPV16 sequence features and severity of cervical disease were limited to AAS E6:L90V which was significantly more common in SCC grade 2/3 than in CINII/III cases (p=0.015). Conclusions: Highly conserved HPV16 genomes circulating in Latvia harbour series of unique, as well as common nonsynonymous SNPs with respective AAS, with one, AAS E6:L90V, associating with disease severity. No HPV vaccine escape variants were detected. Deciphering complete genomes of HPV16 from CIN and SCC cases in Latvia informs public authorities performing HPV vaccination and is useful for management of HPV-associated cervical diseases.