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. 2010 Apr 8;6(4):e1000849.
doi: 10.1371/journal.ppat.1000849.

SARS-CoV pathogenesis is regulated by a STAT1 dependent but a type I, II and III interferon receptor independent mechanism

Matthew B Frieman  1 , Jun Chen, Thomas E Morrison, Alan Whitmore, William Funkhouser, Jerrold M Ward, Elaine W Lamirande, Anjeanette Roberts, Mark Heise, Kanta Subbarao, Ralph S Baric
Affiliations

SARS-CoV pathogenesis is regulated by a STAT1 dependent but a type I, II and III interferon receptor independent mechanism

Matthew B Frieman et al. PLoS Pathog. .

Abstract

Severe acute respiratory syndrome coronavirus (SARS-CoV) infection often caused severe end stage lung disease and organizing phase diffuse alveolar damage, especially in the elderly. The virus-host interactions that governed development of these acute end stage lung diseases and death are unknown. To address this question, we evaluated the role of innate immune signaling in protection from human (Urbani) and a recombinant mouse adapted SARS-CoV, designated rMA15. In contrast to most models of viral pathogenesis, infection of type I, type II or type III interferon knockout mice (129 background) with either Urbani or MA15 viruses resulted in clinical disease outcomes, including transient weight loss, denuding bronchiolitis and alveolar inflammation and recovery, identical to that seen in infection of wildtype mice. This suggests that type I, II and III interferon signaling play minor roles in regulating SARS pathogenesis in mouse models. In contrast, infection of STAT1-/- mice resulted in severe disease, high virus titer, extensive pulmonary lesions and 100% mortality by day 9 and 30 post-infection with rMA15 or Urbani viruses, respectively. Non-lethal in BALB/c mice, Urbani SARS-CoV infection in STAT1-/- mice caused disseminated infection involving the liver, spleen and other tissues after day 9. These findings demonstrated that SARS-CoV pathogenesis is regulated by a STAT1 dependent but type I, II and III interferon receptor independent, mechanism. In contrast to a well documented role in innate immunity, we propose that STAT1 also protects mice via its role as an antagonist of unrestrained cell proliferation.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Mouse adapted SARS-CoV (rMA15) infection of 129 WT, IFNAR1−/−, IFNGR−/− and STAT1−/− mice.
A. 10 week old female 129 WT, IFNAR1−/−, IFNGR−/− and STAT1−/− mice were infected intranasally with 1×105 pfu/ml rMA15 virus. Individual mice were weighed daily and their average weight change from day 0 is presented. 10 mice per timepoint and strain were used in each group. The * =  a p value of .001. B. At days 2, 5 and 9 post-infection, groups of mice described in A were sacrificed and lungs were dissected, homogenized and supernatant used to titer virus in a plaque assay on Vero cells. Plotted is the average pfu/g of lung for 5 mice per group for each timepoint. The #  =  a p value of .005 and the *  =  a p value of .001. Each is a comparison of Day 9 WT compared to Day 9 STAT1−/−.
Figure 2
Figure 2. Histopathology of rMA15 virus infected mouse strains.
Mice were sacrificed at days 2, 5 and 9 post-infection for histopathological analysis. Lung sections were stained with H&E and photomicrographs of individual airways are shown in the figure. The left side of each matched pair is a 10X picture and the right side is 40X.
Figure 3
Figure 3. In situ hybridization of rMA15 infected mouse lungs.
A. Paraffin embedded mouse lungs where sectioned and probed with S35 labeled probe complementary to SARS-CoV nucleocapsid RNA. Representative samples from days 2, 5 and 9 post-infection (dpi) are shown. Not shown are slides probed with a non-specific probe, which showed no labeling at all. Mock infected samples also showed no labeling. B. High magnification image of a 9 day post-infection STAT1−/− mouse lung with a corresponding H&E stained slide next to it. Note that areas with in situ signal overlap with areas of infiltration in the H&E stained section.
Figure 4
Figure 4. Protein and gene expression analysis of cytokines.
A–D. A cytometric bead array was used to quantify the protein levels of cytokines in lung homogenates after infection with the Urbani virus. Lung samples were homogenized and protein levels of (A) TNFα, (B) IFNγ, (C) MCP1 and (D) IL6 were quantified. E–F. Real-time PCR analysis was performed on RNA from infected mouse lungs. Mice were harvested at days 2, 5 and 9 post-infection and lung RNA isolated by Trizol. cDNA was made from 1ug of RNA of each and used in real-time analysis and the results for 3 mice per group were combined. Fold increase over mock-infected lungs of each cytokine is graphed for (E) TNFα, (F) IFNγ, (G) MCP1 and (H) IL6.
Figure 5
Figure 5. Pathogenesis of Urbani and rMA15 viruses in IL28Ra−/− mice.
BALB/c and IL28Ra−/− mice were infected with 1×105 pfu of either Urbani or rMA15 virus. A. Real time analysis of IL28B transcripts was performed on WT 129 infected mice at 2, 5 and 9 days post infection. Graphed is the fold increase over 18S RNA for each mouse with 5 mice at each timepoint averaged together. B. Titer of Urbani and rMA15 virus at each timepoint. Lungs were homogenized and supernatant of each lung was titered by plaque assay on Vero cells. Shown is the average titer from 5 mice at each timepoint. The level of detection is shown by a dotted line at 100 pfu/ml.
Figure 6
Figure 6. IFNL receptor antibody treatment of IFNAR1−/− mice.
IFNAR1−/− mice were treated with 100µg of anti-IL28Ra antibody and infected with rMA15 virus. A. Mock infected (PBS) and antibody injected IFNAR1−/− mice were infected with 1×105 pfu/ml rMA15 virus at day 0. Average weights for each group are plotted compared to starting weights (5 mice per group). B. Mouse lungs were harvested at days 2, 4 and 9 post-infection. Virus titer in lung homogenates were determined on Vero cells. 5 mice were used per timepoint and the average titer of the group of 5 mice is shown.
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