[Construction and verification of infectious clones for human respiratory syncytial virus subgroup A genotype ON1 expressing fluorescent reporter genes].

Objective: To construct a reverse genetic system for the genotype ON1 of human respiratory syncytial virus subtype A (HRSV-A) expressing fluorescent reporter genes. Methods: Recombinant plasmids encoding EGFP or mCherry were constructed based on the 2019 Beijing HRSV-A ON1 dominant strain (6914). Recombinant viruses, rescued by co-transfecting BSR/T7-9 cells with helper plasmids, were identified via indirect immunofluorescence, whole-genome sequencing, and Western blot. Biological properties were characterized through fluorescent quantitative RT-PCR (qRT-PCR), immunostaining plaque assay and fluorescent focus assays (FFA). Results: Two recombinant viruses expressing EGFP or mCherry (rRSVA6914-EGFP and rRSVA6914-mCherry) were successfully rescued. Western blot analysis confirmed that the expression levels of key structural proteins (G, F, and N) in the recombinant strains were consistent with the parental virus. Multistep growth curve analysis revealed that the replication kinetics of the two recombinant viruses in HEp-2 cells did not differ significantly from those of the parental strain. Two recombinant viruses exhibited substantial neutralizing activity against both palivizumab and nirsevimab used in clinical settings. Furthermore, the viral titer of rRSVA6914-mCherry in A549 cells [(1.19±0.05)×105 PFU/ml] was significantly higher than in HEp-2 cells [(7.60±0.79)×104 PFU/ml] (P<0.001). For rRSVA6914-EGFP, the viral titers determined by immunostaining plaque assay and FFA methods were (1.15±0.17)×105 PFU/ml and (1.36±0.19)×105 FFU/ml. For rRSVA6914-mCherry, the corresponding titers were (3.50±0.23)×104 PFU/ml and (3.37±0.07)×104 FFU/ml. There was no statistically significant difference between the immunostaining plaque assay and FFA methods (both P>0.05). Conclusion: The HRSV-A genotype ON1 reverse genetic system expressing fluorescent reporter genes has been successfully constructed and systematically verified, providing a scientific tool for investigating the pathogenic mechanism of genotype ON1 and for screening antiviral drugs.