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Comparative Study
. 2012 Jan;86(2):884-97.
doi: 10.1128/JVI.05957-11. Epub 2011 Nov 9.

Molecular determinants of severe acute respiratory syndrome coronavirus pathogenesis and virulence in young and aged mouse models of human disease

Matthew Frieman  1 , Boyd Yount, Sudhakar Agnihothram, Carly Page, Eric Donaldson, Anjeanette Roberts, Leatrice Vogel, Becky Woodruff, Diana Scorpio, Kanta Subbarao, Ralph S Baric
Affiliations
Comparative Study

Molecular determinants of severe acute respiratory syndrome coronavirus pathogenesis and virulence in young and aged mouse models of human disease

Matthew Frieman et al. J Virol. 2012 Jan.

Abstract

SARS coronavirus (SARS-CoV) causes severe acute respiratory tract disease characterized by diffuse alveolar damage and hyaline membrane formation. This pathology often progresses to acute respiratory distress (such as acute respiratory distress syndrome [ARDS]) and atypical pneumonia in humans, with characteristic age-related mortality rates approaching 50% or more in immunosenescent populations. The molecular basis for the extreme virulence of SARS-CoV remains elusive. Since young and aged (1-year-old) mice do not develop severe clinical disease following infection with wild-type SARS-CoV, a mouse-adapted strain of SARS-CoV (called MA15) was developed and was shown to cause lethal infection in these animals. To understand the genetic contributions to the increased pathogenesis of MA15 in rodents, we used reverse genetics and evaluated the virulence of panels of derivative viruses encoding various combinations of mouse-adapted mutations. We found that mutations in the viral spike (S) glycoprotein and, to a much less rigorous extent, in the nsp9 nonstructural protein, were primarily associated with the acquisition of virulence in young animals. The mutations in S likely increase recognition of the mouse angiotensin-converting enzyme 2 (ACE2) receptor not only in MA15 but also in two additional, independently isolated mouse-adapted SARS-CoVs. In contrast to the findings for young animals, mutations to revert to the wild-type sequence in nsp9 and the S glycoprotein were not sufficient to significantly attenuate the virus compared to other combinations of mouse-adapted mutations in 12-month-old mice. This panel of SARS-CoVs provides novel reagents that we have used to further our understanding of differential, age-related pathogenic mechanisms in mouse models of human disease.

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Figures

Fig 1
Fig 1
rMA15 viruses. (A) Genomic sequence of rMA15 showing the 6 amino acid changes found in the passaged virus. (B) Chart of rMA15 mutant viruses used in the experiments. In the mutant names, a plus sign means that the virus contains the MA15 amino acid at the position identified, and a minus sign means that the virus contains the WT Urbani amino acid at that position. Under the protein designations, MA indicates an rMA15 mutation at the designated allele, while WT indicates an Urbani amino acid at the designated allele. (C) Growth curves of the mutant viruses in Vero E6 cells, showing efficient replication of the recombinant mutant viruses described in panel B.
Fig 2
Fig 2
Pathogenesis of rMA15 mutant viruses in young BALB/c mice. (A) Percentages of weight loss for mice infected with rMA15 variant viruses. MA−N9- and MA−S-infected mice show reductions in weight loss during infection. Ten mice per time point per virus were used. (B) Titers of rMA15 variant viruses at 2 and 4 dpi in the lung. Titers in lung samples were determined on Vero E6 cells. Five experiments were carried out for each virus and time point. (C) Lung sections were stained with H&E and were scored for the amount of inflammation in each section. Scoring was carried out five times for each time point and virus. (D) Images of representative H&E-stained lung sections at 2 and 4 dpi.
Fig 3
Fig 3
rMA15 nsp9 and spike mutants in young mice. (A) Percentages of weight loss for mice infected with the rMA15 variant viruses diagramed in Fig. 1B. Note that young mice infected with the nsp9 or S variant show no weight loss, while those infected with rMA15 do. Five experiments were carried out for each virus. (B) Titers of rMA15 variant viruses in the lungs at 2 and 4 dpi. Titers in lung samples were determined on Vero E6 cells. Note that there was no difference between WT and mutant viruses. Five experiments were carried out for each virus.
Fig 4
Fig 4
Pathogenesis of rMA15 genome variants in aged mice. (A) Chart showing rMA15 variant viruses encoding only the structural mutations or only the replicase mutations. (B) Percentages of weight loss for mice infected with rMA15 variant viruses. Note that the variants are as pathogenic as rMA15 virus in old mice. Five experiments were carried out for each virus and time point. (C) Titers of rMA15 variant viruses in the lung at 2 and 4 dpi. Titers in lung samples were determined on Vero E6 cells. Note that there was no difference in lung virus titers among the variants and the wild type. Five experiments were carried out for each virus and time point. (D) Images of representative H&E-stained lung sections at 2 and 4 dpi. Note the high levels of eosinophil and neutrophil infiltration in the lungs of infected mice.
Fig 5
Fig 5
MA15 S and nsp9 variant viruses in aged mice. (A) Percentages of weight loss for mice infected with MA15 variant viruses. Aged mice infected with the MA−N9 or MA−S virus display weight loss similar to that with rMA15. Five experiments were carried out for each virus and time point. (B) Titers of rMA15 variant viruses in the lung at 2 and 3 dpi. Titers in lung samples were determined on Vero E6 cells. Note that there was little difference in lung titers among the variants and the wild type at either 2 or 3 dpi. Five experiments were carried out for each virus and time point. (C) Images of representative H&E-stained lung sections at 2 and 3 dpi. Notice the high levels of inflammation and infiltration in these lungs. Airways display continued inflammation throughout the infection.
Fig 6
Fig 6
MA15 S and nsp9 combination variant viruses in aged mice. (A) Percentages of weight loss of mice infected with rMA15 variant viruses. Aged mice infected with WT+N9/S or MA−N9/S display weight loss similar to that with rMA15. Five experiments were carried out for each virus. (B) Titers of virus in the lungs at 4 dpi were normal for MA−N9/S and WT+N9, while WT+S and WT+N9/S demonstrated >1-log-lower titers. Five experiments were carried out for each virus and time point. (C) Images of representative H&E-stained lung sections at 4 dpi.
Fig 7
Fig 7
(A to C) Dose-response curves of rMA15 variant viruses in aged mice. WT MA15, as well as WT+N9/S (A), MA−N9/S (B), and WT+S (C), was used to infect aged mice with different doses of virus. (D to F) Survival curves are plotted alongside each dose-response curve. Five experiments were carried out for each virus.
Fig 8
Fig 8
Comparative pathogenesis of three mouse-adapted SARS-CoVs. (A) Schematic of mutation differences among rMA15, MA20, and v2163. (B) Weight loss curves for young mice infected with MA15, MA20, or v2163. Five experiments were carried out for each virus. (C) Virus titers in the lungs of mice infected with rMA15, MA20, or v2163 at 2 and 4 dpi. Five experiments were carried out for each virus and time point.
Fig 9
Fig 9
Structural comparison of mouse-adapted SARS-CoV mutations and ACE2. (A) Interactions between the Urbani RBD (blue) and human ACE2 (green). The residues in the RBD that change during adaptation to mouse ACE2 are highlighted (cyan). The human ACE2 residues that interact with those RBD sites are shown in red. A key electrostatic interaction occurs between D38 and K353 of human ACE2 (weakened in mouse ACE2). (B) Interactions between the Urbani RBD (blue) and mouse ACE2 (tan). The residues in the RBD that change during adaptation to mouse ACE2 are highlighted (cyan). The mouse ACE2 residues that likely interact with those RBD sites are shown in red. (C) Adaptation of MA15 to mACE2. The Y436H change allows MA15 to interact more robustly with mACE2, since Y436H likely increases binding via electrostatic interaction with D38 of mouse ACE2. Purple, MA15 RBD; tan, mouse ACE2; yellow, residue that changed in MA15; red, residues interacting with RBD sites that adapted to mACE2. (D) Adaptation of MA20 to mACE2. The Y442L and N479K adaptations allow these residues to interact with N30, N31, and Q34 of mACE2, likely increasing binding affinity. MA20 K479 can bind to both N30 and N31 of MA20 via polar interactions, and MA25 L442 likely interacts with Q34 of mACE2. Green, MA20 RBD; tan, mouse ACE2; yellow, residue that changed in MA20; red, residues interacting with RBD sites that adapted to mACE2. (E) Adaptation of MA25 to mACE2. As with MA15, the Y436H change likely allowed this residue to interact more robustly with D38 of mACE2, with the K353H difference in mACE2 playing a key role. In addition, the Y442F adaptation removes a hydroxyl group from the binding interface, likely mediating a better fit between MA25 and mACE2. Purple, MA25 RBD; tan, mouse ACE2; yellow, residue that changed in MA20; red, residues interacting with RBD sites that adapted to mACE2.
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