Figure 4

qRT-PCR-based microneutralization of RSV ( qPCR- MN). Vero cells were seeded in 96-well culture plates (15,000 cells per well). On the following day, a two-fold dilution series was prepared from a pooled human immunoglobulin reference standard (designated as RSV-Lot 1) starting from an initial concentration of 1%. The virus inoculum (500 TCID₅₀ per well of RSV-A2 or RSV-B1) was mixed with an equal volume of RSV-Lot 1 dilution and incubated for 1 hour at 37°C. After incubation, the mixture was transferred to the plate of seeded Vero cells. At 24 or 48 hours post-infection, cell lysates were prepared using Bio-Rad SPR and subjected to qRT-PCR analysis. RNA copy numbers were normalized to the mean value obtained from virus-infected control wells in the absence of neutralizing immunoglobulin. The neutralization titer was defined as the reciprocal of the highest dilution factor of RSV-Lot 1 necessary to inhibit the PCR signal by 90% (or below the threshold of 10% of the virus control wells indicated by the dotted line). (A) RSV-A2 neutralization assessed at 24 or 48 hours post-infection (each point represents the mean; n = 3). (B) RSV-B1 neutralization assessed at 24 or 48 hours post-infection (each point represents the mean; n = 3). The individual experimental replicates assessed independently (n = 3) are shown for neutralization experiments with (C) RSV-A2 and (D) RSV-B1. Additional neutralization experiments (E) with RSV-A2 assessed at 24 hours post-infection were also performed with a monoclonal antibody with known specificity to the RSV F protein (1200) as well as rabbit sera generated pre- and post-immunization with RSV-A2 (each point represents the mean with corresponding range; n = 3).