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, 83 (9), 4185-94

A Chimeric A2 Strain of Respiratory Syncytial Virus (RSV) With the Fusion Protein of RSV Strain Line 19 Exhibits Enhanced Viral Load, Mucus, and Airway Dysfunction

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A Chimeric A2 Strain of Respiratory Syncytial Virus (RSV) With the Fusion Protein of RSV Strain Line 19 Exhibits Enhanced Viral Load, Mucus, and Airway Dysfunction

Martin L Moore et al. J Virol.

Abstract

Respiratory syncytial virus (RSV) is the leading cause of respiratory failure and viral death in infants. Abundant airway mucus contributes to airway obstruction in RSV disease. Interleukin-13 (IL-13) is a mediator of pulmonary mucus secretion. It has been shown that infection of BALB/c mice with the RSV line 19 strain but not with the RSV A2 laboratory strain results in lung IL-13 and mucus expression. Here, we sequenced the RSV line 19 genome and compared it to the commonly used A2 and Long strains. There were six amino acid differences between the line 19 strain and both the A2 and Long RSV strains, five of which are in the fusion (F) protein. The Long strain, like the A2 strain, did not induce lung IL-13 and mucus expression in BALB/c mice. We hypothesized that the F protein of RSV line 19 is more mucogenic than the F proteins of A2 and Long. We generated recombinant, F-chimeric RSVs by replacing the F gene of A2 with the F gene of either line 19 or Long. Infection of BALB/c mice with RSV rA2 line 19F resulted in lower alpha interferon lung levels 24 h postinfection, higher lung viral load, higher lung IL-13 levels, greater airway mucin expression levels, and greater airway hyperresponsiveness than infection with rA2-A2F or rA2-LongF. We identified the F protein of RSV line 19 as a factor that plays a role in pulmonary mucin expression in the setting of RSV infection.

Figures

FIG. 1.
FIG. 1.
Lung IL-13 levels in mice infected with RSV strains A2, line 19, and Long. BALB/cJ mice were mock infected or infected with 105 PFU of A2, line 19, or Long RSV (five mice per group). The lungs were harvested 8 days p.i., and IL-13 was quantified by an ELISA. Each symbol represents an individual mouse. The horizontal dotted line depicts the limit of detection. The short horizontal lines show the means for the different groups. *, P < 0.05 comparing line 19 to mock, A2, or Long (ANOVA). Results are representative of three experiments.
FIG. 2.
FIG. 2.
Pulmonary mucin expression induced by RSV strains A2, line 19, and Long. BALB/cJ mice were mock infected or infected with 105 PFU of A2, line 19, or Long RSV. The lungs were harvested 8 days p.i., and sections were stained with PAS. Airways were scored 0 to 4 for PAS positivity (Materials and Methods). Fifteen to 40 airways were scored per mouse. The results of two experiments combined are shown. (A) Examples of airway mucin scores. Bright pink PAS-positive cells are indicated by black arrows. (B) Total numbers of mice and airways scored for each group. The pie charts show the percentage of total airways in each group receiving each mucin score. (C) Percentage of airways with a score of 3 or 4. Each symbol represents one mouse. The mean and standard error of the mean (error bar) are indicated. *, P < 0.05 (ANOVA) comparing line 19 to mock, A2, or Long.
FIG. 3.
FIG. 3.
BALB/cJ mice were mock infected or infected with 105 PFU of RSV line 19 or Long strain. The lungs were harvested 8 days p.i. The left lungs were processed for gob-5 Western blotting. Each lane contains 100 μg of lung homogenate protein from one mouse. The positive control (+ control) is lung homogenate pooled from RSV-infected STAT1-deficient mice which exhibit increased mucus expression compared to BALB/c mice (21). The membrane was probed with anti-gob-5, then stripped, and reprobed with anti-actin as a loading control.
FIG. 4.
FIG. 4.
In vitro growth and in vivo viral load of F-chimeric RSV strains. (A) Infectious yield in supernatants of HEp-2 cells infected at a multiplicity of infection (MOI) of 0.5 with RSV A2, line 19, Long, rA2-A2F, rA2-line19F, and rA2-LongF. Virus titers from cells infected with Long and rA2-LongF were greater than those from cells infected with other viruses (P < 0.05 by ANOVA) as indicated by the asterisks. Error bars represent standard errors of the means for three separate infections. (B) BALB/cJ mice were infected with 105 PFU of rA2-A2F, rA2-line19F, or rA2-LongF (four mice per group). The lungs were harvested at the indicated days p.i., and infectious RSV was titrated by plaque assay. Values for the rA2-line19F-infected group were significantly different (P < 0.05 by ANOVA) from the values for the rA2-A2F- or rA2-LongF-infected group as indicated by the asterisk. Values for the rA2-line19F-infected group were significantly different (P < 0.05 by ANOVA) from the values for the rA2-LongF-infected group as indicated by the dagger symbol. The dotted line represents the limit of detection. Data from two experiments are shown (n = 8 per data point). (C) BALB/cJ mice were infected with 105 PFU of A2, line 19, Long, rA2-A2F, rA2-line19F, or rA2-LongF (five mice per group). The lungs were harvested 4 days p.i., and RSV RNA levels were quantified by qRT-PCR using RSV F-specific primers. Each symbol represents one mouse. Short horizontal lines show the means and standard errors of the means. P < 0.05 by ANOVA, as indicated by an asterisk above a bracket. NS, not significantly different (P > 0.05 by ANOVA).
FIG. 5.
FIG. 5.
Lung IFN-α levels 24 h p.i. (A) BALB/cJ mice were mock infected or infected with 105 PFU of RSV strain A2, line 19, or Long (five mice per group). The lungs were harvested 24 h p.i., and the levels of IFN-α (in picograms per milliliter) were quantified by an ELISA. (B) BALB/cJ mice were mock infected or infected with 105 PFU of rA2-A2, rA2-line19F, or rA2-LongF (five mice per group). The lungs were harvested 24 h p.i., and IFN-α was quantified by an ELISA. Each symbol represents an individual mouse. The horizontal dotted line depicts the limit of detection. The short horizontal lines show the means and standard errors of the means. P values were calculated using ANOVA and Bonferroni posttest. Values that were significantly different (P < 0.05 by ANOVA using Tukey posttest) are indicated by an asterisk above the bracket.
FIG. 6.
FIG. 6.
Lung IL-13 levels in mice infected with rA2-A2, rA2-line19F, or rA2-LongF. BALB/cJ mice were mock infected or infected with 105 PFU of rA2-A2, rA2-line19F, or rA2-LongF (four or five mice per group). The lungs were harvested 8 days p.i., and the level of IL-13 (in picograms per milliliter) was quantified by an ELISA. Each symbol represents an individual mouse. The horizontal dotted line depicts the limit of detection. The short horizontal lines show the means of the different groups. Values for the rA2-line19F-infected group were significantly different (P < 0.05 by ANOVA) from values for the mock-infected and rA2-A2F- and rA2-LongF-infected groups. Results of three experiments combined are shown.
FIG. 7.
FIG. 7.
Pulmonary mucin expression induced by rA2-A2, rA2-line19F, or rA2-LongF. BALB/cJ mice were mock infected or infected with 105 PFU of rA2-A2, rA2-line19F, or rA2-LongF (four or five mice per group). The lungs were harvested 8 days p.i., and sections were stained with PAS. Individual airways were scored 0 to 4 for PAS positivity. Results of two experiments are shown. (A) Total numbers of mice and airways scored for each group. The pie charts show the percentage of airways receiving each mucin score (0 to 4). (B) Percentage of airways with a score of 3 or 4. Each symbol represents one mouse. The means and standard errors of the means are indicated by short horizontal lines. Values for the rA2-line19F-infected group were significantly different (P < 0.05 by ANOVA) from values for the mock-infected and rA2-A2F- or rA2-LongF-infected groups.
FIG. 8.
FIG. 8.
BALB/cJ mice were infected with 105 PFU of A2, line 19, Long, rA2-A2F, rA2-line19F, or 19 or rA-LongF (five mice per group). The lungs were harvested 8 days p.i and processed for gob-5 Western blotting. Each lane contains 100 μg of lung homogenate protein total pooled from five mice. The membrane was probed with anti-gob-5, then stripped, and reprobed with anti-actin as a loading control.
FIG. 9.
FIG. 9.
BALB/cJ mice were mock infected or infected with 105 PFU of rA2-A2, rA2-line19F, or rA2-LongF (five mice per group). AHR was measured 9 days p.i. Results of two experiments are shown (n = 10 per data point). The means ± standard errors of the means (error bars) are shown. Values for the rA2-line19F-infected group were significantly different (P < 0.05 by ANOVA) from values for the mock-, rA2-A2F-, and rA2-LongF-infected groups at a 30-mg/ml methacholine dose as indicated by the asterisk. The values for the 30-mg/ml methacholines were significantly different (P < 0.05 by ANOVA) from the value for the baseline 0-mg/ml methacholine dose as indicated by the dagger symbol.

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