Table 1.
Summary of HPV isolates and sequenced complete genomes.
Figure 1.
Alpha-9 phylogenetic tree showing representative types and variant lineages.
A phylogenetic tree was constructed using the MrBayes (v3.1.2) program [48] inferred from the global alignment of complete circular genome nucleotide sequences linearized at the first ATG of the E1 ORF. To root the tree, HPV34 and HPV73 prototype sequences (NCBI accession numbers NC_001587 and NC_006165, respectively) were set as the outgroup and are represented by grey broken lines. The Bayesian credibility values less than 100 were indicated on or near the branch nodes. The shaded areas represent groupings of lineages and sublineages of HPV16, HPV31, HPV33, HPV35, HPV52, HPV58 and HPV67. The length of broken 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.
Figure 2.
HPV31variant tree topologies andpairwise comparisons of individual complete genomes.
Bayesian trees were inferred from global alignment of complete genome nucleotide sequences (the other HPV16-related HPV reference prototypes were set as the outgroup). Numbers on or near branches indicate support indices in the following order: Bayesian credibility value using MrBayes v3.1.2 [48], maximum parsimony (MP) bootstrap percentage and neighbor joining (NJ) bootstrap percentage using PAUP* v4.0b10 [50]. An asterisk (*) indicates 100% agreement between methods. “NA” reflects disagreement between a method and the reference Bayesian tree at a given node. Thus, one tree is shown, but three different methods of tree construction were used to estimate the support of the provided tree, as explained above. Distinct variant lineages (i.e., termed A, B, and C) are classified according to the topology and nucleotide sequence differences from >1% to <10%. 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 each 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. Symbols and lines used are different for each distinct variant lineage to facilitate visual comparisons. For example, percentage differences of variant lineage A are indicated by X's and connected by broken lines; values for isolates of variant lineage B are indicated by closed triangles and connected by solid lines; and, difference values of lineage C isolates are indicated by open circles and connected by a dotted line.
Figure 3.
HPV33 variant tree topologies and pairwise comparisons of individual complete genomes.
The phylogenetic tree was constructed as described in Figure 2. Distinct variant lineages (i.e., termed A and B) are classified according to the topology and nucleotide sequence differences from >1% to <10%. Distinct sublineages (i.e., termed A1 and A2) were also inferred from the tree topology and nucleotide sequence differences in the >0.5% to <1% range. The percent nucleotide sequence differences were calculated and are shown in the panel to the right of each phylogeny as described in Figure 2.
Figure 4.
HPV35 variant tree topologies and pairwise comparisons of individual complete genomes.
The phylogenetic tree was constructed as described in Figure 2. There were no distinct variant lineages however, sublineages (i.e., termed A1 and A2) were inferred from the tree topology and nucleotide sequence differences in the >0.5% to <1% range. The percent nucleotide sequence differences were calculated and are shown in the panel to the right of each phylogeny as described in Figure 2.
Figure 5.
HPV52 variant tree topologies and pairwise comparisons of individual complete genomes.
The phylogenetic tree was constructed as described in Figure 2. Distinct variant lineages (i.e., termed A, B, C and D) are classified according to the topology and nucleotide sequence differences from >1% to <10%. Distinct sublineages (i.e., termed B1, B2, C1 and C2) were also inferred from the tree topology and nucleotide sequence differences in the >0.5% to <1% range. The percent nucleotide sequence differences were calculated and are shown in the panel to the right of each phylogeny as described in Figure 2.
Figure 6.
HPV58 variant tree topologies and pairwise comparisons of individual complete genomes.
The phylogenetic tree was constructed as described in Figure 2. Distinct variant lineages (i.e., termed A, B, C and D) are classified according to the topology and nucleotide sequence differences from >1% to <10%. Distinct sublineages (i.e., termed A1, A2, A3, B1, B2, D1 and D2) were also inferred from the tree topology and nucleotide sequence differences in the >0.5% to <1% range. The percent nucleotide sequence differences were calculated and are shown in the panel to the right of each phylogeny as described in Figure 2.
Figure 7.
HPV67 variant tree topologies and pairwise comparisons of individual complete genomes.
The phylogenetic tree was constructed as described in Figure 2. Distinct variant lineages (i.e., termed A and B) are classified according to the topology and nucleotide sequence differences from >1% to <10%. Distinct sublineages (i.e., termed A1 and A2) were also inferred from the tree topology and nucleotide sequence differences in the >0.5% to <1% range. The percent nucleotide sequence differences were calculated and are shown in the panel to the right of each phylogeny as described in Figure 2.
Figure 8.
Diagnostic lineage-specific single nucleotide polymorphisms (SNPs) and their position in the genome.
Lineage-specific SNPs were determined from alignments of type specific variants using the program MacClade v4.08 [55]. The position of variants across HPV lineage(s) and sublineage(s) are displayed to the right of the name of the clade from which the data was abstracted, as depicted in the phylogenetic trees in Figures 2–7. The viral genome sequence differences for each sequenced isolate are displayed in Figure S2. Regions of the genome are displayed below the x-axis for reference. The graphic output was generated using Microsoft Excel.
Figure 9.
Single-nucleotide polymorphism (SNP) rarefaction curves.
The program EstimateS v8.2.0 for Mac OS (downloaded from: http://viceroy.eeb.uconn.edu/EstimateS) was used to illustrate the curves. The Y-axis represents the total number of parsim-informative single nucleotide polymorphisms (SNPs) observed in at least 2 genomes of a specific type. Insertion and deletions are counted as one event equal to a single SNP. The X-axis shows the number of sequenced isolates. The curve generated for variants of each HPV type are displayed by different lines as indicated by the key to the right of the curves. For reference, the number of variable nucleotide positions for HPV31, HPV33, HPV35, HPV52, HPV58 and HPV67 genomes are 3.8%, 2.4%, 1.8%, 4.4%, 5.1% and 1.7%, respectively (see Table 1).
Table 2.
Calculation of HPV31, 33 and 35 variations by genome region and open reading frame (ORF).
Table 3.
Calculation of HPV52, 58 and 67 variations by genome region and open reading frame (ORF).