Figure 7.

Recombinant RSV carrying R170T/K172T mutations in M induces higher inflammation and is cleared faster than wild type in a mouse model of RSV infection. BALB/c mice were inoculated with 50 µL of 2.5 × 10⁵ pfu of rA2 or rA2-R170T/K172T or an equivalent volume of diluent on day 0. Five mice from each group were euthanized on the indicated days p.i., and samples collected for analysis. (A) One lung from each animal was put into 1 mL of diluent with steel beads and frozen at 80 °C. Lungs were subsequently homogenized in a Tissue-lyser, debris removed by centrifugation and the supernatant used immediately for plaque assay. Results represent the mean +/− SEM (n = 5) for pfu/lung, representative of two separate experiments. White columns—rA2, black columns—rA2-R170T/K172T. * p < 0.05. (B) The second lung from each animal was fixed in formalin, embedded in paraffin, sectioned, and stained with haemotoxylin and eosin (H&E). Sections were imaged using a Leica DM750 microscope and the LAS software; five images were obtained at each of three different depths from each lung. Representative sections from each experimental group are shown, typical of two separate experiments. (C) Inflammation around bronchi was quantitated independently by two operators according to the schema described in Section 2.16 (0–9 scale [43]) using digital images such as those shown in (A). Quantitation was performed on multiple lung lobes from 3 different depths of sectioned tissue; results represent the mean + SEM (n ≥ 50). Grey columns—diluent, white columns—rA2, black columns—rA2-R170T/D172T. *** p < 0.001. (D) Blood was collected by cardiac puncture. Systemic inflammation was determined by ELISA for RANTES as described. Results represent the mean +/− SEM (n = 5). Grey columns—diluent, white columns—rA2, black columns—rA2-R170T/D172T. * p < 0.05.