Classification and nomenclature system for human Alphapapillomavirus variants: general features, nucleotide landmarks and assignment of HPV6 and HPV11 isolates to variant lineages

Abstract

Background: Papillomaviruses constitute a family of viruses that can be classified into genera, species and types based on their viral genome heterogeneity. Currently circulating infectious human Alphapapillomaviruses (alpha-PVs) constitute a set of viral genomes that have evolved from archaic times and display features of host co-speciation. Viral variants are more recently evolved genomes that require a standardized classification and nomenclature.

Objectives: To describe a system for the classification and nomenclature of HPV viral variants and provide landmarks for the numbering of nucleotide positions.

Methods: The complete 8 kb genomes of the alpha-9 species group and HPV6 and 11 types, collected from isolates throughout the world were obtained from published reports and GenBank. Complete genomes for each HPV type were aligned using the E1 start codon and sequence divergence was calculated by global and pairwise alignments using the MUSCLE program. Phylogenetic trees were constructed from the aligned sequences using a maximum likelihood method (RAxML).

Results: Pairwise comparisons of nucleotide differences between complete genomes of each type from alpha-9 HPV isolates (HPV16, 31, 33, 35, 52, 58 and 67) revealed a trimodal distribution. Maximum heterogeneity for variants within a type varied from 0.6%-2.3%. Nucleotide differences of approximately 1.0%-10.0% and 0.5%-1.0% of the complete genomes were used to define variant lineages and sublineages, respectively. Analysis of 43 HPV6 complete genomes indicated the presence of 2 variant lineages, whereas 32 HPV11 isolates were highly similar and clustered into 2 sublineages. A table was constructed of the human alpha-PV landmark nucleotide sequences for future reference and alignments.

Conclusions: A proposed nomenclature system for viral variants and coordination of nucleotide positions will facilitate the comparison of variants across geographic regions and amongst different populations. In addition, this system will facilitate study of pathogenic, tissue tropism and functional differences amongst variant lineages of and polymorphisms within HPV variants.

Figure 1. Distribution of pairwise differences between nucleotide sequences of alpha-9 type genomes

The genome nucleotide sequences of types 16, 31, 33, 35, 52, 58 and 67, previously reported (8, 20), were globally aligned using the program MUSCLE (21). The p-distance method in MEGA5 (22) was used to calculate the percent differences for each isolate compared to all other isolates of the same type based on a global alignment. The Y-axis represents the number of comparisons. The X-axis shows the percent nucleotide pairwise differences. (A) Comparison of each isolate to all other isolates of the same type, resulting in a total of 3577 assessments; (B) Inter- and intra-lineage pairwise differences. Inter-lineage: comparisons of isolates within different lineages of the same type (2213 comparisons). Intra-lineage: comparisons of isolates within the same lineage (1362 comparisons); (C) Inter-and intra-sublineage pairwise differences. Inter-sublineage: comparisons of isolates within different sublineages of the same lineage (578 comparisons). Intra-sublineage: comparisons of isolates within the same sublineage (784 comparisons).

Figure 2. HPV6 variant tree topology and pairwise comparisons of individual complete genomes

A maximum likelihood (ML) tree was inferred from a global alignment of 43 complete genome nucleotide sequences of HPV6 using RAxML HPC v7.2.8 (23). Distinct variant lineages (i.e., termed A and B) and sublineages (i.e., termed B1, B2 and B3) are classified according to the topology and nucleotide sequence differences from > 1% to < 10%, and > 0.5% to < 1% ranges (4, 8). The percent nucleotide sequence differences were calculated for each isolate compared to all other isolates of the same type based on the complete genome nucleotide sequences and are shown in the panel to the right of the phylogeny. Values for each comparison of a given isolate are connected by lines and the comparison to self is indicated by the 0% difference point.

Figure 3. HPV11 variant tree topologies and pairwise comparisons of individual complete genomes

A ML phylogenetic tree was constructed from 32 HPV11 aligned complete genomes as described in Figure 2. Distinct sublineages (i.e., termed A1 and A2) were inferred from the tree topology and nucleotide sequence differences in the range of ~ 0.5%. The percent nucleotide sequence differences were calculated for each HPV11 isolate compared to all other HPV11 isolates based on the complete genome nucleotide sequences and are shown in the panel to the right of the phylogeny. Values for each comparison of a given isolate are connected by lines and the comparison to self is indicated by the 0% difference point.

Figure 4. Alpha-10 phylgenetic tree showing representative types and variant lineages/sublineages

A maximum likelihood tree was constructed using RAxML HPC v7.2.8 (23) inferred from the global alignment of complete genome nucleotide sequences linearized at the first ATG of the E1 ORF. Representative alpha-8 HPV types, HPV7 (NCBI accession number NC_001595), HPV40 (NC_001589), HPV43 (NC_005349) and HPV91 (NC_004085), were set as the outgroup and are shown by grey dashed lines. The shaded areas represent groupings of lineages and sublineages of HPV6 and HPV11. The length of dashed and solid lines represent distance between clades, although the number of changes is different for these two lines; the scale is indicated in the upper left corner of the figure. The GenBank accession numbers of alpha-10 HPV types are listed in the brackets following each variant: HPV6∣A (X00203), HPV6∣B1 (FR751337), HPV6∣B2 (FR751328), HPV6∣B3 (L41216), HPV11∣A1 (M14119), HPV11∣A2 (FN907962), HPV13∣A (X62843), HPV13∣B (DQ344807), HPV44 (U31788), HPV44s (U31791), HPV74 (AF436130).