In vitro Characterization of rRSVs

The above mentioned immunoplaque assay and RT-qPCR were used to evaluate the replication activity and confirm the successful rescue of the rRSVs since passage 1 (P1) in Vero cells. Simultaneously, a GoScript™ reverse transcription (RT)-PCR kit was used to assess the genetic stability of the rescued rRSVs by amplifying fragments of viral DNA harboring the mutated nucleotides contributing to the att phenotype. The primers used for RT-PCR are shown in Supplementary Table S1. These characteristic DNA fragments were amplified for each rRSV sample from every odd-numbered passage from P1 to P9 and subsequently sequenced to monitor the possible reversion of individual point mutations.

The rRSV growth kinetics was also assayed using a previously described method (Collins and Bermingham 1999; Schickli et al. 2012). Briefly, HEp-2 cells were infected with wtRSV or rRSVs at a multiplicity of infection (MOI) of 0.1 in triplicate and incubated at 32 °C. Cells and supernatants were harvested at 24-h intervals post-infection, and viral titers were determined by immunoplaque assay as mentioned above.

The temperature-sensitive phenotype of rRSVs in vitro was evaluated by determining the efficiency of plaque formation at various temperatures, through a modified method from a previous report (Crowe et al. 1993). Briefly, a HEp-2 cell monolayer, grown in 24-well plates, was inoculated with tenfold serial dilutions of wtRSV and rRSV cultures, incubated for 5 days at 32 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, and 40 °C, and then assayed for infectivity at each corresponding temperature using the immunoplaque assay.

The temperature stress test was performed as reported previously (Collins and Bermingham 1999; Schickli et al. 2012). Briefly, rRSVs were passaged at elevated non-permissive temperatures, twice at 37 °C, twice at 39 °C, and once at 40 °C. Next, HEp-2 cells were inoculated with rRSVs at 0.1 MOI in a 96-well plate. Following a 1-h incubation at 33 °C, the inoculum was collected by pipetting, and cells were supplied with 200 μL/well of DMEM with 2% FBS and incubated at 37 °C. Following a 5-day incubation at 37 °C, 100 μL of the media from each of the infected wells were transferred to an uninfected 96-well plate containing HEp-2 cells, in duplicate. After a 1-h incubation, the media were removed by pipetting and the cells were supplied with 200 μL/well of fresh medium. The plates were then incubated for 5 days at 37 °C for the second passage. The virus was similarly transferred and incubated at 39 °C to generate the third and fourth passages. The last passage was performed at 40 °C. For each passage, one of the duplicate HEp-2 plates was immunostained to assess RSV infectivity.