Differential pathogenesis of respiratory syncytial virus clinical isolates in BALB/c mice - PubMed (original) (raw)

. 2011 Jun;85(12):5782-93.

doi: 10.1128/JVI.01693-10. Epub 2011 Apr 6.

Michael H Chi, Kaori Sakamoto, Dawn C Newcomb, Michael G Currier, Matthew M Huckabee, Sujin Lee, Kasia Goleniewska, Carla Pretto, John V Williams, Anne Hotard, Taylor P Sherrill, R Stokes Peebles Jr, Martin L Moore

Affiliations

Differential pathogenesis of respiratory syncytial virus clinical isolates in BALB/c mice

Kate L Stokes et al. J Virol. 2011 Jun.

Abstract

Airway mucus is a hallmark of respiratory syncytial virus (RSV) lower respiratory tract illness. Laboratory RSV strains differentially induce airway mucus production in mice. Here, we tested the hypothesis that RSV strains differ in pathogenesis by screening six low-passage RSV clinical isolates for mucogenicity and virulence in BALB/cJ mice. The RSV clinical isolates induced variable disease severity, lung interleukin-13 (IL-13) levels, and gob-5 levels in BALB/cJ mice. We chose two of these clinical isolates for further study. Infection of BALB/cJ mice with RSV A2001/2-20 (2-20) resulted in greater disease severity, higher lung IL-13 levels, and higher lung gob-5 levels than infection with RSV strains A2, line 19, Long, and A2001/3-12 (3-12). Like the line 19 RSV strain, the 2-20 clinical isolate induced airway mucin expression in BALB/cJ mice. The 2-20 and 3-12 RSV clinical isolates had higher lung viral loads than laboratory RSV strains at 1 day postinfection (p.i.). This increased viral load correlated with higher viral antigen levels in the bronchiolar epithelium and greater histopathologic changes at 1 day p.i. The A2 RSV strain had the highest peak viral load at day 4 p.i. RSV 2-20 infection caused epithelial desquamation, bronchiolitis, airway hyperresponsiveness, and increased breathing effort in BALB/cJ mice. We found that RSV clinical isolates induce variable pathogenesis in mice, and we established a mouse model of clinical isolate strain-dependent RSV pathogenesis that recapitulates key features of RSV disease.

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Figures

Fig. 1.

Fig. 1.

Differential weight loss patterns with RSV clinical isolates. (A) BALB/cJ mice were mock infected (n = 4) or infected with 5 × 105 PFU of A2, 2-20, or 3-12 (n = 8/group). (B) BALB/cJ mice were mock infected (n = 6) or infected with 105 PFU of A2 (n = 8), 2-20 (n = 10), 12-35 (n = 10), 3-12 (n = 10), line 19 (n = 8), or Long (n = 6). Weight loss ± standard error of the mean is shown. *, at day 2, values for RSV 2-20 and 3-12 were significantly lower (P < 0.05, ANOVA) than those of other RSV strains; †, at day 6, values for RSV 2-20 and 12-35 were significantly lower (P < 0.05, ANOVA) than those of other RSV strains.

Fig. 2.

Fig. 2.

Weight loss after infection with UV-inactivated RSV 2-20. BALB/cJ mice were mock infected (n = 8), infected with 1.2 × 106 PFU of 2-20 (n = 8), or infected with UV-inactivated 2-20 (n = 8). Weight loss ± standard error of the mean is shown.

Fig. 3.

Fig. 3.

BALB/cJ mice were mock infected (n = 3) or infected with 105 PFU of the indicated RSV strain (n = 4/group). IL-13 protein levels were quantified by ELISA in left lung lobe homogenates. The dashed line represents the limit of detection. *, P < 0.05 compared to mock; †, significantly higher (P < 0.05, ANOVA) than line 19 infection.

Fig. 4.

Fig. 4.

gob-5 Western blotting. BALB/cJ mice were mock infected with 105 PFU of RSV strains A2, line 19, and Long or clinical isolate strains (n = 5/group). Lungs were harvested at 8 days p.i. Each lane contains 100 μg of total lung homogenate protein pooled from five mice. The membrane was probed with anti-gob-5 and then stripped and reprobed with anti-actin as a loading control.

Fig. 5.

Fig. 5.

In vitro growth of RSV strains A2, 2-20, and 3-12. Infectious yield in supernatants of HEp-2 cells (A) and BEAS-2B cells (B) infected at an MOI of 0.5 with RSV A2, 2-20, or 3-12. Error bars represent standard error of the mean of three separate infections. *, titers from cells infected with RSV 3-12 are significantly less than titers from cells infected with A2 or 2-20 (P < 0.05 ANOVA).

Fig. 6.

Fig. 6.

Viral load of RSV strains A2, 2-20, and 3-12. BALB/cJ mice were infected with 5 × 105 PFU of A2, 2-20, or 3-12 (n = 5/group). Lungs were harvested at the indicated days p.i., and infectious RSV was titrated by an immunodetection plaque assay. Data are means ± standard error of the mean. *, at day 1 p.i. values for 2-20 and 3-12 were significantly higher than those for A2 and line 19 (P < 0.05, ANOVA); **, at day 4 p.i. the value for A2 was significantly higher than values for 2-20, 3-12, and line 19 (P < 0.05, ANOVA); †, at day 6, value for A2 was significantly higher than values for 2-20, 3-12, and line 19 (P < 0.05, ANOVA). The dotted line represents the limit of detection.

Fig. 7.

Fig. 7.

RSV antigen in bronchiolar epithelium. BALB/cJ mice were infected with 5 × 105 PFU of A2, 2-20, or 3-12 (n = 5/group). Lungs harvested at day 1 p.i. were probed for RSV by immunofluorescence as described in Materials and Methods. The left column shows RSV-positive cells (fluorescein isothiocyanate [green] staining) in RSV A2-, 2-20-, and 3-12-infected mice and nuclei (DAPI blue counterstain). White arrowheads indicate FITC-positive cells. The right column (H&E) depicts lung sections adjacent to those in the left column, showing airways corresponding to those in the immunofluorescence images. Black arrowheads indicate necrotic cell debris. Scale bar, 50 μm.

Fig. 8.

Fig. 8.

Early histopathologic lesions of RSV 2-20 and 3-12 infection. BALB/cJ mice were mock infected (A) or infected with 5 × 105 PFU of A2 (B), 2-20 (C), or 3-12 (D) (n = 4 to 5 per group). Lungs were harvested at 1 day p.i., stained with H&E, and analyzed for histologic changes as described in Materials and Methods. Representative airways are shown. Yellow bars indicate distance between vascular wall and adjacent tissue as a measure of perivascular edema. Arrows indicate intrabronchiolar exudates of necrotic cell debris. Scale bar, 50 μm.

Fig. 9.

Fig. 9.

RSV A2001/2-20 induced high levels of pulmonary mucin expression. BALB/cJ mice were mock infected (n = 3 per group) or infected with 105 PFU of A2 (n = 4), line 19 (n = 4), 2-20 (n = 5), or 3-12 (n = 5). Lungs were harvested at 8 days p.i. and processed for PAS staining. The lung tissues were digitized, and each area of airway epithelium was annotated by hand as described in Materials and Methods. (A) Example of airway epithelium (indicated by a thin red line) in 2-20-infected mouse. The software detects PAS positivity based on color (thin red lines outlining PAS stain). (B) The percentage of area that was PAS positive for each airway was determined; >200 individual airways are shown per group. Each round symbol represents one airway. Capped lines indicate significant differences between groups (P < 0.05, ANOVA). (C) Same percent PAS data as in panel B plotted against the size of all airways. The airway depicted in panel A is indicated by an arrow.

Fig. 10.

Fig. 10.

RSV 2-20-induced mucin expression is IL-13-dependent. BALB/cJ mice or IL-13 knockout (KO) mice were mock-infected (n = 2 to 4) or infected with 105 PFU of 2-20 (n = 7 to 10 per group). Lungs were harvested at 8 days p.i. and processed for PAS staining. The lung tissues were digitized, and each area of airway epithelium was annotated by hand as described in Materials and Methods. Percent PAS positivity is plotted per group. Capped lines indicated significant differences between groups (P < 0.001, ANOVA).

Fig. 11.

Fig. 11.

Lung dysfunction caused by RSV 2-20. (A) BALB/cJ mice were mock infected (n = 4) or infected with 105 PFU of A2, 2-20, or 3-12 RSV. Breath distension of peripheral arteries, a measure of pulsus paradoxus and pulmonary obstruction, was quantified at the indicated days noninvasively by pulse oximetry as described in Materials and Methods. *, P < 0.05 comparing 2-20 to mock, A2, and 3-12 groups. (B) BALB/cJ mice were mock infected or infected with 105 PFU of 2-20 or line 19 (n = 10/group). AHR was measured at 9 days p.i. Means and standard errors of the means are shown. *, P < 0.05 comparing 2-20 or line 19 to mock at 100 mg/ml methacholine dose.

Fig. 12.

Fig. 12.

RSV A2 and 3-12 induced higher levels of IFN-γ-producing CD8+ T cells than 2-20 and line 19. BALB/cJ mice (n = 5 per group) were mock infected or infected with 105 PFU of A2, line 19, 2-20, or 3-12. Lungs were harvested at 8 days p.i., and cells were isolated using a Ficoll gradient. A total of 1 × 106 cells were stained with fluorescently conjugated anti-CD3, anti-CD8, and anti-IFN-γ antibodies and then analyzed by flow cytometry. Total cells were calculated as described in Materials and Methods. Each symbol represents one mouse. Means and standard errors of the means are shown for each group. Brackets indicate significant differences between groups (P < 0.05, ANOVA).

Fig. 13.

Fig. 13.

IFN-γ-producing CD8+ T cells in 2-20-infected BALB/cJ and IL-13 knockout (KO) mice. IL-13 KO mice (n = 5 for mock; n = 9 per group for RSV infection) or wt BALB/cJ mice were mock infected (n = 5) or infected with 105 PFU of 2-20 (n = 8). Lungs were harvested at day 8 p.i., and cells were isolated using a Ficoll gradient. Cells were stained with fluorescently conjugated anti-CD3, anti-CD8, and anti IFN-γ antibodies and then analyzed by flow cytometry. Total cells were calculated as described in Materials and Methods. Means and standard errors of the means are show for each group.

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