Effect of Ribavirin and Glucocorticoid Treatment in a Mouse Model of Human Metapneumovirus Infection

Abstract

Human metapneumovirus (hMPV)-infected BALB/c mice were treated with ribavirin (40 mg/kg of body weight twice a day intraperitoneally), corticosterone (0.2 mg/ml in water), or both modalities. Ribavirin significantly decreased both hMPV replication in lungs (by 5 log₁₀) and global pulmonary inflammation on day 5 postinfection, whereas glucocorticoids reduced only alveolar and interstitial inflammation, compared to controls.

Human metapneumovirus (hMPV) is a newly described member of the Paramyxoviridae family associated with acute respiratory tract infections in all age groups, with more-severe diseases occurring in young children, elderly individuals, and immunocompromised hosts (3, 7, 8, 15). Experimental animal models of hMPV infection have been reported for primates and rodents (9, 12, 17, 19). Notably, the BALB/c mouse has been described as a good and convenient animal model for this virus due to efficient viral replication and significant lung inflammation associated with systemic and respiratory symptoms when a high intranasal inoculum is used (1, 9). Although ribavirin has been reported to decrease hMPV titers in vitro (18), this drug has not been evaluated with animal models. In this study, we assessed the effects of ribavirin with or without glucocorticoids on hMPV replication and pulmonary inflammation by using a previously described BALB/c mouse model (9).

Animal studies.

Forty-eight 4- to 6-week-old BALB/c mice (Charles Rivers Laboratories, Wilmington, MA) were randomized into six groups of eight mice each, based on weight. On day 0, four groups (32 mice) were infected intranasally with 1 × 10⁸ 50% tissue culture infective doses (TCID₅₀) of hMPV strain C-85473 (type A) at passage 7 in 25 μl of hMPV infection medium as previously reported (9). The other two groups (16 mice) were sham infected with infection medium, and all animals were housed in groups of four in microisolator cages. Groups of hMPV-infected mice were treated with phosphate-buffered saline (PBS), ribavirin (MP Biomedicals, Aurora, OH) at 40 mg/kg of body weight every 12 h, corticosterone (Sigma, St. Louis, MO) at 0.2 mg/ml, or ribavirin and corticosterone. Groups of sham-infected mice were treated with either PBS or ribavirin as described above. Ribavirin and PBS were given intraperitoneally from day 0 to day 5 postinfection, beginning 12 h postinfection, whereas corticosterone was given in water ad libitum from day 3 to day 5. Mice were weighted every day and then sacrificed on day 5 postinfection. The right lungs were homogenized in infection medium and used to determine viral titers in LLC-MK2 cells, whereas the left lungs were used for determination of histopathological scores based on a scale previously described (9, 14).

Effects of ribavirin and corticosterone on hMPV infection.

hMPV-infected mice treated with PBS gradually lost 16.3% ± 6.81% of their initial weight until day 5 postinfection, whereas sham-infected mice receiving PBS gained 4.3% ± 1.15% and controls treated with ribavirin lost 3.7% ± 3.27% (Fig. 1). hMPV-infected mice treated with ribavirin or corticosterone stopped losing weight on day 2 to day 3 postinfection (mean weight loss of 10.8% ± 7.76% and 7.7% ± 5.87% on day 5, respectively), whereas the group treated with both ribavirin and corticosterone gained weight gradually from day 3 to day 5, with a mean weight loss of 6.1% ± 3.48% on day 5. Apart from sham-infected mice, the group that received both treatment modalities was the only one with a statistically significant increase in weight (P < 0.05) on day 5 compared to untreated hMPV-infected mice.

FIG. 1.

Weights of hMPV- and sham-infected mice. The weights of hMPV- and sham-infected mice treated with PBS, ribavirin, glucocorticoids (gluco), or both ribavirin and glucocorticoids were evaluated on a daily basis to observe the effects of the different treatments. *, statistically significant differences (P < 0.05) in weight loss between these groups and hMPV-infected mice receiving PBS, based on the Mann-Whitney rank sum test, were observed. BID, twice a day.

Viral loads were significantly decreased (P < 0.05) in hMPV-infected mice treated with ribavirin and both ribavirin and corticosterone compared to viral loads in mice treated with PBS, with mean viral titers of 4.34 × 10², 1.25 × 10², and 1.12 × 10⁷ TCID₅₀/g of lungs, respectively (Fig. 2). No decrease in lung viral titers was observed for the group treated with corticosterone only. The mean histopathological scores (which are the total scores based on the sum of peribronchial, perivascular, interstitial, and alveolar inflammation) were also significantly decreased (P < 0.05) for lungs of infected mice treated with ribavirin (score of 4.85 ± 1.48) and both ribavirin and glucocorticoids (score of 3.57 ± 0.90) compared to scores for untreated infected mice (score of 10.13 ± 2.1) (Fig. 3A). As previously described (9), the histopathological changes in untreated mice consisted predominantly of alveolar and interstitial inflammation, with some peribronchiolitis and perivasculitis (Fig. 3B). Glucocorticoids alone did not significantly reduce the total histopathological score (8.14 ± 3.44) compared to that for untreated infected mice but significantly decreased interstitial and alveolar inflammation (P < 0.05). The combination of glucocorticoid and ribavirin treatment added modest but nonsignificant improvement of histopathology compared to ribavirin alone.

FIG. 2.

Mean viral titers in lungs of hMPV-infected mice. On day 5 postinfection, right lungs from eight hMPV- or sham-infected mice per group were removed and homogenized to determine mean viral titers expressed as TCID₅₀ per gram of lung. *, statistically significant differences (P < 0.05) between these groups and hMPV-infected mice receiving PBS, based on the Mann-Whitney rank sum test, were observed. Gluco, glucocorticoids.

FIG. 3.

Lung inflammation in hMPV-infected mice. On day 5 postinfection, left lungs from eight hMPV- or sham-infected mice per group were removed and fixed with 10% formalin. Thin sections of paraffin-embedded lung tissues were cut and stained with hematoxylin and eosin. (A) Degrees of lung inflammation (mean histopathological scores) were evaluated for peribronchial, perivascular, interstitial, and alveolar areas. The histopathological score was defined as previously described (9, 14). Statistically significant differences (P < 0.05) in mean total histopathological scores (*) and in interstitial and alveolar inflammation (#) between these groups and hMPV-infected mice receiving PBS, based on the Mann-Whitney rank sum test, were observed. Gluco, glucocorticoids. (B) A representative lung section (×10 enlargement) is shown for each group.

Our study demonstrates that intraperitoneal ribavirin decreases both hMPV replication (mean reduction in viral titers of 5 log₁₀, i.e., from 10⁷ to 10² TCID₅₀/g) and pulmonary inflammation in BALB/c mice. Treatment of mice with glucocorticoids (corticosterone) only significantly reduces some types of pulmonary inflammation (interstitial and alveolar), whereas the combination of the two modalities seems to have a modest additional effect on weight loss and lung inflammation in hMPV-infected mice.

Ribavirin is a broad-spectrum nucleoside analogue with activity against paramyxoviruses, such as human respiratory syncytial virus (hRSV), pneumonia virus of mice, and measles virus, when evaluated with different animal models (10, 20). It has also recently been reported to exhibit similar antiviral activities against hRSV and hMPV when evaluated in vitro with LLC-MK2 cells (18). In our study, early ribavirin treatment for 4 days, starting 12 h postinfection, drastically reduced hMPV replication in lungs of BALB/c mice, with a parallel decrease in pulmonary inflammation. Whether ribavirin treatment could also decrease the possible long-term consequences (e.g., bronchoconstriction and airway hyperresponsiveness) associated with some paramyxovirus infections in mouse models (11), as it has been reported to do for infants with severe hRSV bronchiolitis (5), needs further evaluation. Also, the potential toxic effects related to high-dose ribavirin therapy should be carefully assessed before its clinical use.

Glucocorticoids are powerful suppressors of the innate immune response and were found to be effective in reducing the severity of symptoms associated with some paramyxovirus infections, mainly those caused by parainfluenza viruses (2, 6). However, their efficacy in the treatment of hRSV infections remains controversial (4, 13, 16). As expected, glucocorticoids were ineffective in decreasing the hMPV viral load, but they were more beneficial in reducing weight loss and lung inflammation, suggesting that such immunomodulatory agents could have a place along with potent antiviral therapy for severe hMPV infections. Notably, we started glucocorticoids on day 3 postinfection to allow some initial immune response and recruitment of inflammatory cells before blocking a potentially detrimental inflammatory response as reported with hRSV (13). Other more potent and long-lasting glucocorticoids, such as dexamethasone, as well as various administration routes and schedules (early and delayed therapy) should also be studied, with an emphasis on evaluation of pulmonary functions of mice by use of a plethysmograph. In summary, both compounds tested in our study but particularly ribavirin had benefits on different aspects of hMPV-induced pathology and should be considered for further evaluation with other animal models (such as the cotton rat) and with other modes of delivery (e.g., aerosolized therapy).