Towards a unified classification for human respiratory syncytial virus genotypes

Abstract

Since the first human respiratory syncytial virus (HRSV) genotype classification in 1998, inconsistent conclusions have been drawn regarding the criteria that define HRSV genotypes and their nomenclature, challenging data comparisons between research groups. In this study, we aim to unify the field of HRSV genotype classification by reviewing the different methods that have been used in the past to define HRSV genotypes and by proposing a new classification procedure, based on well-established phylogenetic methods. All available complete HRSV genomes (>12,000 bp) were downloaded from GenBank and divided into the two subgroups: HRSV-A and HRSV-B. From whole-genome alignments, the regions that correspond to the open reading frame of the glycoprotein G and the second hypervariable region (HVR2) of the ectodomain were extracted. In the resulting partial alignments, the phylogenetic signal within each fragment was assessed. Maximum likelihood phylogenetic trees were reconstructed using the complete genome alignments. Patristic distances were calculated between all pairs of tips in the phylogenetic tree and summarized as a density plot in order to determine a cutoff value at the lowest point following the major distance peak. Our data show that neither the HVR2 fragment nor the G gene contains sufficient phylogenetic signal to perform reliable phylogenetic reconstruction. Therefore, whole-genome alignments were used to determine HRSV genotypes. We define a genotype using the following criteria: a bootstrap support of $\ge$ 70 per cent for the respective clade and a maximum patristic distance between all members of the clade of ≤0.018 substitutions per site for HRSV-A or ≤0.026 substitutions per site for HRSV-B. By applying this definition, we distinguish twenty-three genotypes within subtype HRSV-A and six genotypes within subtype HRSV-B. Applying the genotype criteria on subsampled data sets confirmed the robustness of the method.

Keywords: classification; genotypes; human respiratory syncytial virus.

Figure 1.

Genome structure of HRSV. The HRSV-B reference genome (AF013254) is 15,225 nt long and comprises ten genes, coding for eleven proteins.

Figure 2.

Whole-genome phylogenies and density distributions of patristic distances for HRSV-A and HRSV-B. Patristic distances were calculated between all tips of the ML trees of whole-genome alignments of HRSV-A (A) and HRSV-B (C) and cut-off values were chosen at the lowest point after the major peak in the density plot, determined at 0.018 subst./site for HRSV-A (B) and slightly higher at 0.026 subst./site for HRSV-B (D).

Figure 3.

Genotypes defined within subtypes HRSV-A and HRSV-B. Based on the genotype criteria of BS $\ge$ 70 per cent, PatDist_max ≤0.018 subst./site (HRSV-A), or ≤0.026 subst./site (HRSV-B), we distinguish twenty-three and six genotypes for subtype HRSV-A (A) and HRSV-B (B), respectively. Underneath each tree, the evolutionary distance scale is indicated, expressed as nt subst./site.

Figure 4.

Genotype co-occurrence matrix HRSV-A. The heat map shows for each genotype defined using the full data set (y axis), the percentage of taxa that corresponds to the genotypes defined using a subset of the data set (x axis). The correlation of the taxa is 100 per cent for all genotypes, with the exception of the taxa within genotypes A22 and A23 that are merged into one genotype when using a subset of the data.