Circulation of new lineages of RSV-A and RSV-B in Kuwait shows high diversity in the N- and O-linked glycosylation sites in the G protein between 2020 and 2022

Abstract

The human respiratory syncytial virus (RSV) is a significant health concern, particularly for infants, young children, and the elderly. This virus is known to evolve continuously due to environmental factors and herd immunity. In light of this, our study aimed to analyze the genetic variability of the G protein in RSV-A and RSV-B genotypes in Kuwait from 2020 to 2022. Between January 2020 and September 2022, we collected 490 respiratory samples from hospitalized patients with acute respiratory tract infections. These samples were tested and confirmed positive for RSV using multiplex Real-Time PCR. Subsequently, the samples underwent nucleic acid sequencing using the advanced Nanopore sequencing technology to analyze the full-length G gene. Sequence analysis showed that 64 isolates (76%) were RSV-A, and 20 isolates (24%) were RSV-B. The G genes of RSV-A belonged to genotype GA2.3.5, while all the RSV-B genotypes belonged to GB5.0.5a. New lineages and sub-lineages of RSV-A and RSV-B were detected, indicating the circulation of new strains in Kuwait. Many unique and new amino acid changes, including insertions, were found in the G proteins of Kuwaiti isolates, with the highest variability in the second hypervariable region. An increased number of N and O-linked glycosylation sites were also identified in the G protein, which could speculate to alter the antigenicity of RSV. The identified changes in the G protein of RSV-A and RSV-B genotypes might result from immune pressure and could affect the antigenic characteristics of circulating strains in Kuwait. This could potentially lead to new RSV variants that can evade the immune response. Our in-depth analysis of the G proteins of both RSV-A and RSV-B could aid in the development of more potent treatments and vaccines.

Keywords: G protein; Kuwait; glycosylation; nanopore sequencing; respiratory syncytial virus.

Figure 1

Phylogenetic tree of RSV-A (A) and RSV-B (B) in Kuwait. The phylogenetic trees G protein gene was constructed using the Maximum Likelihood method and General Time Reversible/JTT matrix-based models. Kuwaiti strains are blue, and reference strains are black. The scale bar indicates the proportion of nucleotide substitutions, and the branch nodes show bootstrap values with marron dotes. (A) The phylogenetic tree of RSV-A genotypes (GA2.3.5 sub-type) in Kuwait is divided into two lineages: lineage 1 is shaded in green, and lineage 2 is shaded in blue. (B) The phylogenetic tree of the RSV-B genotype (GB5.05.a subtype) in Kuwait is divided into two lineages: lineage 1 is shaded in green, and lineage 2 is shaded in blue.

Figure 2

Shanon entropy plots of deduced amino acid sequences of G protein. (A) RSV-A, n=64) (B) RSV-B, n=20) (C) all Kuwaiti strains (N=84) with their respective prototype strains. The threshold value was set at 0.2. Amino acid sites with entropy values <0.2 are considered conserved, and values >0.2 are deemed viable. The 13 aa, aa 164-176, conserved among all strains, is in orange boxes. The Lowes panel shows aa sequences that include CCD, the CX3C motif (aa 182-186), and the HBD (aa 197-198). CCD, central conserved domain.

Figure 3

Descriptive alignment of the G protein of RSV-A and B genotypes circulated in Kuwait. (A) alignment of RSV-A genotype with MH760605/GA2 prototype strain (B) alignment of RSV-B genotype with MN163124/GB5 prototype strain. Dots display identical residues. Stop codons are displayed by asterisks. Molecular markers are indicated in grey shading. The potential N-glycosylation sites (NXT, where X is not proline) are indicated by blue dots. The potential sites for extensive O-glycosylation are not shown.

Figure 4

(A) Deduced amino acid alignment and mutations in the second hyper-variable region of the G protein (227-298) of RSV-A genotype. The Kuwaiti strains were aligned with the MH760605/GA2 prototype strain. Dots display identical residues. Stop codons are indicated by asterisks. (B) mutations in the amino acids of the Kuwaiti strains. An asterisk shows the stop codon in the prototype strain. Amino acid in red is inserted AA in the Kuwaiti strains.

Figure 5

(A) Deduced amino acid alignment and mutations in the second hyper-variable region of the G protein (227-299) of RSV-B genotype. The Kuwaiti strains were aligned with the MN163124/GB5 prototype strain. Dots display identical residues. Stop codons are indicated by asterisks. (B) mutations in the amino acids of the Kuwaiti strains. An asterisk shows the stop codon in the prototype strain. Amino acids in red are inserted AA in the Kuwaiti strains.