Identification of distinct genotypes in circulating RSV A strains based on variants in the virus replication-associated genes

Abstract

Respiratory syncytial virus (RSV) is a common cause of respiratory infection that often leads to hospitalization of infected younger children and older adults. RSV is classified into two strains, A and B, each with several subgroups or genotypes. One issue with the definition of these subgroups is the lack of a unified method of identification or genotyping. We propose that genotyping strategies based on the genes coding for replication-associated proteins could provide critical information on the replication capacity of the distinct subgroups, while clearly distinguishing genotypes. Here, we analyzed the virus replication-associated genes N, P, M2, and L from de novo assembled RSV A sequences obtained from 31 newly sequenced samples from hospitalized patients in Philadelphia and 78 additional publicly available sequences from different geographic locations within the United States. In-depth analysis and annotation of variants in the replication-associated proteins identified the polymerase protein L as a robust target for genotyping RSV subgroups. Importantly, our analysis revealed non-synonymous variations in L that were consistently accompanied by conserved changes in its co-factor P or the M2-2 protein, suggesting associations and interactions between specific domains of these proteins. Similar associations were seen among sequences of the related human metapneumovirus. These results highlight L as an alternative to other RSV genotyping targets and demonstrate the value of in-depth analyses and annotations of RSV sequences as it can serve as a foundation for subsequent in vitro and clinical studies on the efficiency of the polymerase and fitness of different virus isolates.IMPORTANCEGiven the historical heterogeneity of respiratory syncytial virus (RSV) and the disease it causes, there is a need to understand the properties of the circulating RSV strains each season. This information would benefit from an informative and consensus method of genotyping the virus. Here, we carried out a variant analysis that shows a pattern of specific variations among the replication-associated genes of RSV A across different seasons. Interestingly, these variation patterns, which were also seen in human metapneumovirus sequences, point to previously defined interactions of domains within these genes, suggesting co-variation in the replication-associated genes. Our results also suggest a genotyping strategy that can prove to be particularly important in understanding the genotype-phenotype correlation in the era of RSV vaccination, where selective pressure on the virus to evolve is anticipated. More importantly, the categorization of pneumoviruses based on these patterns may be of prognostic value.

Keywords: genotypes; polymerase L; replication-associated genes; respiratory syncytial virus.

Fig 1

Summary of genetic variations of RSV A 11 CDS regions. Each point represents the number of variants per sample, N = 109. (A) Total variations per sample for each CDS region. (B) Non-synonymous variations per sample for each CDS region. One-way analysis of variance (ANOVA) non-parametric Kruskal-Wallis tests were performed for statistical significance between CDS regions. ns P > 0.1234; *P > 0.03; ****P < 0.0001. Comparisons between all the CDS regions to G or L were **** unless otherwise noted by a line between CDS regions.

Fig 2

Principal component analysis (PCA) of the 109 RSV A sequences analyzed in this study. Plots outlined in squares are shown as a comparison between our predicted groups and other genotyping methods. Data points (circles) in each plot were labeled based on (A) predicted groups, (B) location in the United States, (C) year the sample was collected, (D) Nextstrain clades classification, and (E) Goya clades classification. Each data point represents a sequence. The distance between two data points on the plot depicts the similarity between the sequences. Hence, a cluster of data points means that those sequences are of similar clade phylogenetically. The percentage of variance in all the plots are PC1 = 58.71%, PC2 = 21.78%, and PC4 = 2.52%. Abbreviations in the “LOCATION” legend indicate the state the sequence was collected from. UNKN is used when a US state was not assigned to the sequence.

Fig 3

Maximum likelihood (ML) phylogeny tree of 109 RSV A (A) full-length sequences and (B) CDS region of G. Color bars represent assigned genotypes based on the GA clades and our predicted groups (R1–R6). The trees were rooted on reference sequence—KT992094.1. Dotted lines were used to shorten the distance between the reference and the rest of the sequence for easier visualization.

Fig 4

Annotation of non-synonymous variations observed within the domains of RSV A replication-associated genes for each predicted R group. The top illustration is the full-length genome of RSV with colored regions depicting the replication-associated genes. The illustrations below are zoomed in on the domains of the replication-associated genes. All variations shown in each predicted group, i.e., R1–R5 were seen at least three times along with other variations within the same predicted group. Areas shaded gray along R1–R5 indicate consistent domains with variations. R6 was omitted as it had no variation present in any of the domains. Abbreviation key as follows: NTD, N-terminal domain; CTD, C-terminal domain; OD, oligomerization domain; ZBD, zinc-binding domain; M2-1 CD, core domain; RdRP, RNA-dependent RNA polymerase; Cap, cap addition domain; L CD, connector domain; MT, cap methylation domain.