Genomic characterization of circulating human respiratory syncytial viruses A and B in Kuwait using whole-genome sequencing

Abstract

The human respiratory syncytial virus (RSV) is considered one of the most common viruses that infect children globally. The virus is known to have extensive gene sequence variability within and between RSV groups A and B globally; however, there is no information on the whole-genome characterization and diversity of RSV in Kuwait. Therefore, this study aimed to sequence the entire genome of RSV strains isolated from patients with acute respiratory tract infection (ARTI) in Kuwait. Therefore, this study aimed to sequence the entire genome of RSV strains isolated from patients with ARTI in Kuwait. Between January 2020 and September 2022, 7,093 respiratory samples were collected from hospitalized infants, children, and adults and were analyzed for respiratory viruses by multiplex real-time PCR. Whole-genome sequencing using the Oxford Nanopore sequencing technology was performed on 84 RSV-positive samples. The results revealed a higher prevalence of group A (76%) than group B (24%) RSV isolates. Phylogenetic analysis showed that RSV-A strains clustered with the GA2.3.5 sub-genotype and RSV-B strains clustered with the GB5.0.5a sub-genotype; however, forming new lineages of RSV-A and RSV-B circulated in Kuwait during this period. Genetic variability was higher among the group A viruses than group B viruses, and the rate of synonymous and missense mutations was high in genes other than the G protein-coding gene. We also detected several known and unique molecular markers in different protein-coding genes. This is the first study in Kuwait to characterize the whole genomes of RSV A and B to identify the circulating genotypes, comprehend the genetic diversity and the evolution of the virus, and identify important genetic markers associated with specific genotypes.IMPORTANCEWhole-genome sequencing of respiratory syncytial virus (RSV) strains in Kuwait using MinION Nanopore technology was used to characterize and analyze the genotypes and sub-genotypes of the RSV circulating among patients with acute respiratory tract infections in Kuwait. This study also identified known and unknown gene mutations and imported genetic markers associated with specific genotypes. These results will assist in establishing a framework for RSV classification and allow for a better consideration of the mechanisms leading to the generation of diversity of RSV. In addition, these data will allow a comparison of vaccine viruses with those in Kuwait, providing useful insights into future vaccine and therapy strategies for RSV in Kuwait.

Keywords: Kuwait; nanopore sequencing; respiratory syncytial virus; whole-genome sequencing.

Fig 1

Age distribution of RSV-positive patients in Kuwait (%) (n = 490).

Fig 2

Seasonal trends of RSV in Kuwait from January 2020 to September 2022.

Fig 3

RSV-A genotype (n = 64) assignment according to Nextstrain classification from 2020 to 2022. Different colors denote different genotypes and sub-genotypes of RSV-A. Kuwaiti strains are green dots.

Fig 4

RSV-B genotype (n = 20) assignment according to Nextstrain classification from 2020 to 2022. Different colors denote different genotypes and sub-genotypes of RSV-B. Kuwaiti strains are in orange dots.

Fig 5

Phylogenetic analysis of RSV-A strain (A) and RSV-B (B) circulating in Kuwait, January 2020–September 2022. Whole-genome RSV-A and RSV-B sequences from Kuwaiti and reference strain sequences were used to generate a phylogenetic tree using the maximum likelihood method and general time reversible/JTT matrix-based models. Kuwaiti strains are in bold. The scale bar indicates the proportion of nucleotide substitutions, and the numbers at the branches are bootstrap values determined for 1,000 repetitions.

Fig 6

Time-scaled phylogenetic tree of RSV. (A) A tree representing 960 high-coverage genomes of RSV-A from 27 countries sampled between December 1977 and September 2022, including 64 RSV-A isolates from Kuwait. (B) A tree representing 888 high-coverage genomes of RSV-B from 27 countries sampled between July 1977 and September 2022, including 20 RSV-B isolates from Kuwait. The phylogeny was estimated using IQTree under the GTR substitution model and visualized with auspice. The tree was rooted with the RSV-A reference strain AY911262 and RSV-B reference strain NC_001781.

Fig 7

Maximum likelihood IQ TREE of 960 RSV-A (A) and 888 RSV-B (B) genomes from different countries retrieved from the GenBank and the Kuwaiti strains. Kuwaiti strains are in blue branches.

Fig 8

Heatmap demonstrates the percentage of molecular markers in each gene among RSV-A and RSV-B genotypes detected in Kuwait. The color scale indicates the significance of the correlation, with blue and white colors indicating the highest and lowest correlation, respectively.