Reverse genetics systems for contemporary isolates of respiratory syncytial virus enable rapid evaluation of antibody escape mutants

Abstract

Human respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory infection in children under 5 y of age. In the absence of a safe and effective vaccine and with limited options for therapeutic interventions, uncontrolled epidemics of RSV occur annually worldwide. Existing RSV reverse genetics systems have been predominantly based on older laboratory-adapted strains such as A2 or Long. These strains are not representative of currently circulating genotypes and have a convoluted passage history, complicating their use in studies on molecular determinants of viral pathogenesis and intervention strategies. In this study, we have generated reverse genetics systems for clinical isolates of RSV-A (ON1, 0594 strain) and RSV-B (BA9, 9671 strain) in which the full-length complementary DNA (cDNA) copy of the viral antigenome is cloned into a bacterial artificial chromosome (BAC). Additional recombinant (r) RSVs were rescued expressing enhanced green fluorescent protein (EGFP), mScarlet, or NanoLuc luciferase from an additional transcription unit inserted between the P and M genes. Mutations in antigenic site II of the F protein conferring escape from palivizumab neutralization (K272E, K272Q, S275L) were investigated using quantitative cell-fusion assays and rRSVs via the use of BAC recombineering protocols. These mutations enabled RSV-A and -B to escape palivizumab neutralization but had differential impacts on cell-to-cell fusion, as the S275L mutation resulted in an almost-complete ablation of syncytium formation. These reverse genetics systems will facilitate future cross-validation efficacy studies of novel RSV therapeutic intervention strategies and investigations into viral and host factors necessary for virus entry and cell-to-cell spread.

Molecular Modeling Predicts Novel Antibody Escape Mutations in the Respiratory Syncytial Virus Fusion Glycoprotein

“…However, the predominant circulating RSV A strain, ON1, was first identified in 2006 in Ontario and was classified as a novel genotype based off both F gene and G gene sequencing (53). The use of strains that reflect current circulating strains will help produce data that are more clinically relevant and can be done in the same reverse genetics system used for these experiments (54). Molecular modeling is also limited to predictions at individual amino acid residues.…”

Establishment of a new reverse genetics system for respiratory syncytial virus under the control of RNA polymerase II

“…The T7RNAP recovery method has been applied in many previous studies on the RG system of RSV, because this method has been well established for the rescue of recombinant RSV. 10,[15][16][17][18][19][20][21][22][23]37 However, the T7RNAP-driven method has limitations with respect to the use of available cell lines. For example, the stable expression of T7RNAP is required in rescue cells, such as BSR-T7/5 cells.…”

Unprecedented outbreak of respiratory syncytial virus in Taiwan associated with ON1 variant emergence between 2010 and 2020

“…However, the novel ON1 variant in 2020 did not show higher replication or virulence compared with those of a previously circulating genotype in our in vitro and in vivo models. These results need to be confirmed using more suitable model such as reverse genetics and primary cell in the future [ 24 ]. We hypothesized that RSV epidemic is related to antigenic variation of the 2020 novel ON1 variant, which is worth further investigating in the future.…”