Next-generation sequencing of human respiratory syncytial virus subgroups A and B genomes

Human respiratory syncytial virus (HRSV) is a leading cause of acute respiratory illness in young children worldwide. Whole genome sequencing of HRSV offers enhanced resolution of strain variability for epidemiological surveillance and provides genomic information essential for antiviral and vaccine development. A 10-amplicon one-step RT-PCR assay and a 20-amplicon nested RT-PCR assay with enhanced sensitivity were developed to amplify whole HRSV genomes from samples containing high and low viral loads, respectively. Ninety-six HRSV-positive samples comprised of 58 clinical specimens and 38 virus isolates with C t values ≤ 24 were amplified successfully using the 10-amplicon one-step RT-PCR method and multiplexed in a single MiSeq run. Genome coverage exceeded 99.3% for all 96 samples. The 20-amplicon nested RT-PCR NGS method was used to generate >99.6% HRSV full-length genome for 72 clinical specimens with C t values ranging from 24 to 33. Phylogenetic analysis of the genome sequences obtained from the 130 clinical specimens revealed a wide diversity of HRSV genotypes demonstrating methodologic robustness.