Molecular Evolution of Attachment Glycoprotein (G) and Fusion Protein (F) Genes of Respiratory Syncytial Virus ON1 and BA9 Strains in Xiamen, China

Abstract

Monitoring viral transmission and analyzing the genetic diversity of a virus are imperative to better understand its evolutionary history and the mechanism driving its evolution and spread. Especially, effective monitoring of key antigenic mutations and immune escape variants caused by these mutations has great scientific importance. Thus, to further understand the molecular evolutionary dynamics of respiratory syncytial virus (RSV) circulating in China, we analyzed nasopharyngeal swab specimens derived from hospitalized children ≤5 years old with acute respiratory tract infections (ARIs) in Xiamen during 2016 to 2019. We found that infants under 6 months of age (52.0%) were the main population with RSV infection. The prevalent pattern "BBAA" of RSV was observed during the epidemic seasons. RSV ON1 and BA9 genotypes were the dominant circulating strains in Xiamen. Interestingly, we observed four Xiamen-specific amino acid substitution combinations in the G protein and several amino acid mutations primarily occurring at antigenic sites Ø and V in the F protein. Our analyses suggest that introduction of new viruses and local evolution are shaping the diversification of RSV strains in Xiamen. This study provides new insights on the evolution and spread of the ON1 and BA9 genotypes at local and global scales. IMPORTANCE Monitoring the amino acid diversity of the RSV G and F genes helps us to find the novel genotypes, key antigenic mutations affecting antigenicity, or neutralizing antibody-resistant variants produced by natural evolution. In this study, we analyzed the molecular evolution of G and F genes from RSV strains circulating in Xiamen, China. These data provide new insights on local and global transmission and could inform the development of control measures for RSV infections.

Keywords: RSV; attachment glycoprotein; fusion protein; hospitalized children; molecular epidemiology.

FIG 1

Time-scaled MCC trees for G protein of Xiamen RSV A and B strains and reference strains representing known genotypes. The branches of Xiamen ON1 (A) and BA9 (B) are colored red and blue, respectively, and then shown individually alongside in the graph. Four representative sequences from Xiamen ON1 and BA9 strains collected from successive epidemic seasons are labeled by red stars. Scale bars represent the unit of time (year).

FIG 2

ML trees of Xiamen ON1 (A) and BA9 (B) strains were constructed by IQ-TREE. Branches of different subgenotypes are highlighted by different colors. Each subgenotype name is listed above the colored line. Each lineage is marked by color shading. Scale bar shows patristic distance by ranges of 0.01 for RSV A and 0.005 for RSV B. Ultrafast bootstrap values of each ancestral lineage node as statistical support are displayed on the trees.

FIG 3

Characteristic substitution combinations of the G protein ectodomain of Xiamen ON1 and BA9 sublineages. (A) Schematic of the RSV G protein. The RSV A2 G protein (298 amino acids) includes cytoplasmic and transmembrane regions (TM), two hypervariable regions (HVR1 and HVR2), a central conserved domain (CCD), and a highly basic heparin-binding domain (HBD). (B and C) Alignment of deduced amino acid sequences of the G protein ectodomain of Xiamen ON1 (aa 68 to 320) and BA9 (aa 68 to 310) strains relative to the reference strains ON1 (JN257693) and BA9 (DQ227395). The representative sequences marked by red stars from each lineage were aligned with the reference strains for ON1 and BA9 according to Fig. 2A and B. Color shading highlights the unique amino acid variations of different lineages.

FIG 4

Polymorphism analysis of the G protein ectodomain of Xiamen ON1 and BA9 strains. (A) Plots of amino acid variation frequency by position of the G protein ectodomain (A, aa 68 to 320; B, aa 68 to 310) to the reference strains ON1 (JN257693) and BA9 (DQ227395). Red, RSV A; blue, RSV B. Compared with the references, variations at a frequency of >10% (dashed lines) are labeled. (B) Sequence alignment of the sequence repeats in the G protein HRV2 of the ON1 and BA9 strains. The sequence repeats (aa 261 to 283 and 285 to 307 for RSV A; aa 240 to 259 and 260 to 279 for RSV B) are labeled duplication #1 and duplication #2. Motifs used for the analysis are marked by gray shading. Clustering was performed according to the sequences in the duplicated regions. Blank spaces indicate similar amino acids to the references. The percentages of each representative cluster for the ON1 and BA9 strains are listed in the table.

FIG 5

ML trees for G protein of Xiamen RSV A and B strains and global circulating strains (2010 to 2019). ML trees for RSV A (A) and B (B) strains were constructed using the ML method with IQ-TREE and displayed in circular format. Branches of different lineages are highlighted by different colors (red for lineage 1, green for lineage 2, purple for lineage 3, and blue for lineage 4). Xiamen strains obtained in this study are marked by colored dots. Guangdong, Russia, and America strains are labeled by red, blue, and green stars, respectively, in lineage 3 in the ML tree for RSV A strains. Similarly, Switzerland (a green star), Japan (a red star), Australia (purple stars), and Russia (orange stars) strains are labeled by colored stars in lineage 4 in the ML tree for RSV B strains.

FIG 6

Polymorphisms in the F protein of Xiamen RSV A and B strains. Plots of amino acid variation frequency by position of the F protein (aa 26 to 574) to the reference sequences RSV A2 (KT992094) and 9320 (AY353550). Red, RSV A; blue, RSV B. Compared with the references, variations at a frequency of >10% (dashed lines) are labeled.

FIG 7

Variations in neutralizing epitopes of the RSV F protein. The crystal structures of RSV prefusion and postfusion F are displayed according to the PDB files 5W23 and 3RRR, respectively. Neutralizing epitopes (Ø, I, II, III, IV, and V) were surface color coded (deep salmon, Ø; light blue, I; pale yellow, II; pale green, III; light pink, IV; light orange, V). Only amino acids with mutations of >10.0% are highlighted in black on the 3D structures of the F proteins.