Respiratory syncytial virus A genotype classification based on systematic intergenotypic and intragenotypic sequence analysis

Abstract

Respiratory syncytial virus (RSV), a leading cause of lower respiratory tract infections, is classified in two major groups (A and B) with multiple genotypes within them. Continuous changes in spatiotemporal distribution of RSV genotypes have been recorded since the identification of this virus. However, there are no established criteria for genotype definition, which affects the understanding of viral evolution, immunity, and development of vaccines. We conducted a phylogenetic analysis of 4,353 RSV-A G gene ectodomain sequences, and used 1,103 complete genome sequences to analyze the totallity of RSV-A genes. Intra- and intergenotype p-distance analysis and identification of molecular markers associated to specific genotypes were performed. Our results indicate that previously reported genotypes can be classified into nine distinct genotypes: GA1-GA7, SAA1, and NA1. We propose the analysis of the G gene ectodomain with a wide set of reference sequences of all genotypes for an accurate genotype identification.

Figure 1

Phylogenetic analysis of RSV group A. Maximum Likelihood Cladogram of 2,151 unique nucleotide sequences of the G gene ectodomain retrieved from GenBank. Clades are colored according to the genotype assignment.

Figure 2

Phylogenetic analysis of RSV group A. Maximum Clade Credibility (MCC) Tree from Bayesian analysis of 2,151 unique nucleotide sequences of the G gene ectodomain retrieved from GenBank. Clades are colored according to the genotype assignment.

Figure 3

Proportional (p) nucleotide distances for the G gene calculated between and within RSV-A genotypes. Red font underlined p-distances indicates values below intragenotypic threshold distance (0.037) used for clustering sequences in the same genotype. (a) RSV-A distinct genotypes established using a stepwise closest neighbor joining strategy; the NA2 genotype was regrouped within GA2 genotype, and the NA3 and ON1 genotypes were regrouped within NA1 genotype. (b) RSV-A G gene ectodomain Maximum Clade Credibility (MCC) Tree showed that the GA2 genotype includes five distinct subclades (3c and 3d).

Figure 4

Proportional (p) nucleotide distances for the G gene calculated between and within RSV-A genotypes including GA2.1-GA2.5 clades separately. Red font underlined p-distances indicates values below intragenotypic threshold distance (0.037) used for clustering sequences in the same genotype. (a) RSV-A distinct genotypes established using a stepwise closest neighbor joining strategy; the NA4 genotype and GA2.5 subclade were regrouped with the GA3 genotype; the NA2 genotype and GA2.2, GA2.3, and GA2.4 were grouped together and assigned as the GA2 genotype; the NA3 genotype, GA2.1 subclade, and ON1 genotype grouped together with the NA1 genotype. (b) RSV-A G gene ectodomain Maximum Clade Credibility (MCC) Tree showing the clades that conform the new assignment of GA3, GA2, and NA1 genotypes (c).

Figure 5

Phylogenetic analysis of RSV group A. Maximum Clade Credibility (MCC) Tree from Bayesian analysis of 2,151 unique nucleotide sequences of the G gene ectodomain retrieved from GenBank. Clades defined after genotype reassignment, based on p-distance grouping are shown in (a). Period of time (years) of circulation of each genotype based on the date of collection of samples from which the sequences were derived (b).

Figure 6

Phylogenetic analysis of RSV group A. Maximum Likelihood Trees for NS1, NS2, N, P, M, SH, G, F, and M2 genes. (a) Proportional (p) nucleotide distances calculated between and within RSV-A genotypes for each gene. (b) Values in black squares indicate the lowest intragenotype distance for each gene; gray shaded values indicate intergenotype p-distances that are lower than the intragenotype threshold established for each gene.

Figure 7

Distribution of molecular markers present in RSV-A genotypes. The location of all (a) and unique (b) molecular markers present in each genotype is shown.

Figure 8

RSV-A genotype distribution before 1980 and each decade thereafter, according to continent of viral detection.