Analysis of circulating respiratory syncytial virus A strains in Shanghai, China identified a new and increasingly prevalent lineage within the dominant ON1 genotype

Abstract

Respiratory syncytial virus A (RSV-A) is one of the commonest pathogens causing acute respiratory tract infections in infants and children globally. The currently dominant circulating genotype of RSV-A, ON1, was first detected in Shanghai, China in 2011, but little data are available regarding its subsequent circulation and clinical impact here. In this work, we analyzed RSV-A infection in a cohort of patients hospitalized for acute respiratory infections in Shanghai Children's Hospital, and RSV-A was detected in ~10% of these cases. RSV-A G gene sequencing revealed that all successfully sequenced strains belonged to ON1 genotype, but in phylogenetic analysis, the majority of these sequences formed a clade separate from the four previously established lineages within ON1. The new lineage, denoted ON1-5, was supported by phylogenetic analyses using additional G gene sequences from RSV-A strains isolated in Shanghai and elsewhere. ON1-5 first appeared in 2015 in China and the Netherlands, and has since spread to multiple continents and gained dominance in Asia. In our cohort, ON1-5 was not associated with markedly different clinical presentations compared to other ON1 lineages. ON1-5 strains are characterized by four amino acid variations in the two mucin-like regions of G protein, and one variation (N178G) within the highly conserved CCD domain that is involved in receptor binding. These data highlight the continuous evolution of RSV-A, and suggest the possibility of the virus acquiring variations in domains traditionally considered to be conserved for fitness gain.

Keywords: CCD; RSV-A; mucin-like region; phylogenetic analysis; virus evolution.

Figure 1

Respiratory syncytial virus A (RSV-A) and RSV-B infections in children admitted to Shanghai Children’s Hospital with respiratory tract infections, January 2019–March 2020. Hospitalized patients meeting the criteria detailed in the section “Materials and Methods” of the main text were enrolled, and lower respiratory tract aspirates were collected and used for detection of common respiratory tract infection pathogens. Numbers of RSV-A-positive, RSV-B-positive, and all tested cases are plotted against date of illness onset.

Figure 2

Phylogenetic analysis of G gene sequences of RSV-A strains circulating in Shanghai, January 2019–March 2020. RSV-A G sequences obtained from RSV-A-positive samples in this work along with reference strain sequences were used to generate a phylogenetic tree using Randomized Axelerated Maximum Likelihood (RAxML) with 1,000 independent bootstrap searches. Average pairwise distances between ON1-5 strains and other ON1 lineages were calculated using Molecular Evolutionary Genetic Analysis (MEGA).

Figure 3

Phylogenetic analysis of the G gene sequences from RSV-A strains circulating in Shanghai, February 2011–March 2020. (A) RSV-A G sequences obtained from RSV-A-positive samples in this work, derived from archived RSV-A-positive samples collected in previous years, and sequences from strains isolated in Shanghai available from GenBank were used to generate a phylogenetic tree using RAxML with 1,000 independent bootstrap searches along with reference strain sequences. (B) Sequences analyzed in (A) were grouped according to isolation date into epidemic seasons as indicated, and circulating genotypes and lineages were tabulated and plotted for each season.

Figure 4

Circulation of ON1 lineages in different geolocations, January 2019–March 2021. (A) RSV-A G sequences obtained from RSV-A-positive samples in this work, and sequences from strains isolated from other geolocations between January 2019 and March 2021 available from GenBank were used to generate a phylogenetic tree using RAxML for genotype and lineage assignation. Sequences were grouped by location of isolation, and genotype and lineages were tabulated and plotted for each indicated geolocation. (B) Average pairwise distances between ON1–5 strains and other ON1 lineages were calculated using MEGA.

Figure 5

Phylogenetic analysis of all available G gene sequences of RSV-A ON1-5 strains with info on time and location of isolation. RSV-A G sequences obtained from RSV-A-positive samples in this work, and sequences with info on time and location of isolation available from GenBank were used to generate a phylogenetic tree using RAxML for genotype and lineage assignation. All ON1-5 sequences were then used to generate a phylogenetic tree using RAxML as shown here. Small branches of interest are highlighted with magnification on the right. The large red star denotes the first ON1-1 strain isolated in Ontario, Canada (JN257693).

Figure 6

ON1-5-specific amino acid variations in G protein. (A) Schematic representation of ON1-5 G protein. Structural and functional features, as well as ON1-5-specific amino acid variations are shown. (B,C) Amino acid sequences of strains belonging to different ON1 lineages were aligned in MEGA, and regions containing lineage-specific variations are shown. Variations specific to each lineage are highlighted using colored boxes.