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The fusion (F) protein of respiratory syncytial virus (RSV) is the major target of immunoprophylactic monoclonal antibodies (mAbs) and vaccines. Recently reported mutations in F gene antigenic sites can vary among RSV types A and B. To further understand mutations in RSV F proteins, we performed subtyping and F gene sequencing on 400 RSV-positive respiratory samples collected at four pediatric hospitals within the United States from children under 2 years old between 2018 and 2020. RSV B was predominant in 2018-2019 and RSV A in 2019-2020 (55.5% and 85.5% respectively). Compared to the reference sequence, all RSV B samples had at least one antigenic polymorphism with the most changes at sites AM14/V (100%) and Ø (93.3%) followed by II (5.8%), IV (3.9%), and p27 (2.9%). The most frequent mutations among RSV B for AM14/V site were in L172Q (100%), S173L (100%), and K191R (95.2%) while for Ø site they were in I206M (93.3%) and Q209R (93.3%). Conversely, polymorphisms were observed in only 15.3% of RSV A samples overall, specifically at antigenic sites p27 (5.9%), IV (3.0%), II (2.5%), AM14/V (2.0%), I (2.0%), and Ø (0.5%). Among RSV A cases, T122A at p27 (n = 10) and S276N at II (n = 3) were the most common substitution sites. S276N at site II was found in both RSV types. Although polymorphisms in F proteins of RSV B were more common than those in RSV A samples, changes in both subtypes were observed in key F antigenic sites which could potentially impact the efficacy of mAb therapies and vaccines.

Acute viral respiratory infections are a major health burden in children worldwide. In recent years, rapid and sensitive multiplex nucleic acid amplification tests (NAATs) have replaced conventional methods for routine virus detection in the clinical laboratory.We compared BioFire® FilmArray® Respiratory Panel (FilmArray V1.7), Luminex NxTag® Respiratory Pathogen Panel (NxTag RPP) and Applied Biosystems TaqMan Array Card (TAC) for the detection of eight viruses in pediatric respiratory specimens. Results from the three platforms were analyzed with a single-plex real-time RT-PCR (rRT-PCR) assay for each virus.Of the 170/210 single-plex virus-positive samples, FilmArray detected a virus in 166 (97.6%), TAC in 163 (95.8%) and NxTag RPP in 160 (94.1%) samples. The Positive Percent Agreement (PPA) of FilmArray, NxTag RPP and TAC was highest for influenza B (100%, 100% and 95.2% respectively) and lowest for seasonal coronaviruses on both FilmArray (90.2%) and NxTag RPP (81.8%), and for parainfluenza viruses 1- 4 on TAC (84%). The Negative Percent Agreement (NPA) was lowest for rhinovirus/enterovirus (92.9%, 96.7% and 97.3%) on FilmArray, NxTag RPP and TAC respectively. NPA for all three platforms was highest (100%) for both parainfluenza viruses 1- 4 and influenza A and B, and 100% for human metapneumovirus with TAC as well.All three multiplex platforms displayed high overall agreement (gt;90%) and high NPA (gt;90%), while PPA was pathogen dependent and varied among platforms; high PPA (gt;90%) was observed for FilmArray for all eight viruses, TAC for six viruses and NxTag RPP for 4 viruses.