Herbst, K. W. | bioRxiv Science

bioRxiv2026

Mutations and predicted glycosylation patterns in respiratory syncytial virus isolates correlate with disease severity.

Respiratory syncytial virus (RSV) remains an important cause of lower respiratory tract infections in young children, producing mild to life-threatening disease. Although rapid viral evolution through genetic drift is well established, the structural and functional impacts of specific pathoadaptive mutations linked to enhanced virulence are poorly defined. We investigated these relationships by isolating RSV from available nasal swabs of five hospitalized infants during the 2022-2023 winter season and conducting comparative viral genomic analysis. Severity of disease was evaluated using a validated clinical scoring system. Whole-genome sequencing followed by reference-guided assembly and structural modeling revealed distinct amino acid polymorphisms correlating with disease severity. Phylogenetic analysis placed all isolates within the RSV-A GA2.3.5 G clade. Isolates from mild moderate and severe cases clustered in A.D.1.5 and A.D.1.8 subclades. Nineteen amino acid differences were associated with clinical severity and isolates from moderate or severe cases replicated more rapidly in vitro than mild isolates. Computational glycosylation predictions indicated an increasing number of glycosylation sites in the G protein corresponding with greater disease severity. Together, these data suggest that specific pathoadaptive mutations may contribute to enhanced viral replication and severity, and are relevant for future surveillance efforts and the development of immune-based strategies targeting virulence-associated residues.

microbiology↗

bioRxiv2025

Comparison of a long-read amplicon sequencing approach to short-read amplicons for microbiome analysis

Most microbiome studies to date rely on sequencing short amplicons of the 16S rRNA gene on Illuminas platforms. Because of the short read length, sequences often can be identified reliably only to the family or genus levels. Long read sequencing with whole-length 16S rRNA sequencing can improve taxonomic resolution, but often only to the species level. StrainID is an alternative approach that amplifies a large segment of the ribosomal operon, including the entire 16S rRNA gene, internal transcribed spacer, and a portion of the 23S rRNA gene. This longer amplicon is designed to allow ribotype-level classification. Although studies have demonstrated the utility of StrainID for several sample types, it has not yet been validated for saliva. Here, we compared the performance of StrainID to short read amplicons with saliva samples as well as a synthetic mock DNA community and human and mouse fecal samples. Short reads were amplified with primer pairs appropriate for the corresponding sample type, and were classified with two different taxonomic databases. For both saliva and fecal samples, we found that StrainID performed similarly to short reads overall and demonstrated a key benefit with phylogenetic-based beta diversity tests and taxonomic classification. Our results further build on establishing StrainID as a valid method and specifically validate its use with saliva samples. ImportanceThe interpretation of microbiome composition studies is highly dependent on the methodologies chosen during experimental design, which affects factors such as resolution, throughput, cost, and accuracy. StrainID is an approach that can improve resolution while maintaining high-throughput and similar costs to short-read sequencing. The salivary microbiome represents a diverse community of microbes with links to a variety of health conditions and disease states. Closely related strains of bacteria can have drastically different effects on their host. Establishing StrainID as a valid approach for studying the salivary microbiome opens avenues for research that improve upon alternative methods by increasing sensitivity and accuracy compared to traditional short read approaches.

microbiology↗