Fig 1.
Cartoon of RSV G protein motifs and attachment mechanisms.
(A) Domains of RSV G in linear form. (B) Predicted general structure of the RSV G and the likely relative position of its domains. (C) Attachment mechanism of RSV G in immortalized cells and HBE. CT–cytoplasmic tail. TM–transmembrane domain. CCD/noose–central conserved domain/cysteine noose. HBD–heparin binding domain.
Fig 2.
RSV G in laboratory and clinical isolate viruses grown in HEp-2 and HBE.
At 5 dpi, RSV was collected from HEp-2 medium (A) or an apical wash of HBE (B), pelleted by centrifugation and analyzed by gel electrophoresis with SDS and 10% BME and immunoblotted with mAb L9 against G. Subgroup A: A2, 2–20, 3–12 (Lanes 2–4). Subgroup B: B1, 2.32, 894 (Lanes 5–7).
Fig 3.
HBE grown RSV is significantly more infectious for HBE than for HEp-2 cells.
HEp-2 and HBE were inoculated with 300 ffu (based on HEp-2 titration) of RSV-A2(HEp-2) and RSV-A2(HBE). Images of fluorescent cells after 24 h for RSV-A2(HEp-2) on HEp-2 (A) and on HBE (B). Images of fluorescent cells after 24 h for RSV-A2(HBE) on HEp-2 (C) and on HBE (D). Quantified infected cells of each virus inoculated with 100 ffu (based on HEp-2 titration) on HEp-2 and HBE (E). Relative infectivity was calculated by dividing the titer on HBE by that on HEp-2: HBE/HEp-2, mean values in Table 1 (F). Each virus stock was titrated on both HEp-2 and HBE on the same day, inoculating for 2 h at 37°C and counting fluorescent cells at 20 or 24 hpi, respectively. The mean and standard deviation (SD) of three independent experiments are plotted for F. (* p < 0.05, *** p < 0.001, **** p < 0.0001).
Table 1.
Average HBE/HEp-2 values plotted in Fig 3F.
Fold difference was calculated by dividing HBE/HEp-2 value for HBE grown virus, by that of HEp-2 grown virus.
Fig 4.
HBE-grown RSV virions are less infectious on HEp-2 cells when the comparison is based on genome numbers in the inoculum.
Images of HEp-2 and HBE 24 h after inoculation of 107 genomes of RSV(HEp-2) (A and B) and RSV(HBE) (C and D). Infectivity was determined by ffu per 106 genomes on HEp-2 (E) and HBE (D). Viruses were titrated on both cultures to quantify ffu and RNA was extracted from the unused inoculum and genomes quantified by RT-qPCR for the RSV N (nucleocapsid) gene. The mean of three independent experiments are plotted. (*** p < 0.001). Comparison of Ct value when using negative sense specific versus random primers for reverse transcription (G).
Fig 5.
GAG dependency of RSV(HEp-2), RSV(HBE) and RSV-ΔG(HEp-2).
RSV A2 and ΔG viruses grown on HEp-2 or HBE) were titrated on CHO-K1 and CHO-A745. A representative titration experiment (A). The number of foci on CHO-K1 was divided by that on CHO-A745 to calculate the GAG dependency index from three independent experiments (B). (* p < 0.05).
Fig 6.
Infectivity of original and HBE grown clinical RSV samples on HEp-2 and HBE cultures.
Clinical samples were passaged on HBE cultures once to produce HBE grown viruses. A) HBE/HEp-2 ratio based on titration of nasal wash samples or HBE grown virus on HBE and HEp-2 cells. B) HEp-2 ffu per 106 genomes and C) HBE ffu per 106 genomes, based on infectious virus titration and RT-qPCR analysis from the same inoculum. D) Immunoblot of G of the virus produced from HBE cultures infected with each of the clinical nasal wash or nasopharyngeal samples.