A mouse-adapted SARS-coronavirus causes disease and mortality in BALB/c mice

Abstract

No single animal model for severe acute respiratory syndrome (SARS) reproduces all aspects of the human disease. Young inbred mice support SARS-coronavirus (SARS-CoV) replication in the respiratory tract and are available in sufficient numbers for statistical evaluation. They are relatively inexpensive and easily accessible, but their use in SARS research is limited because they do not develop illness following infection. Older (12- to 14-mo-old) BALB/c mice develop clinical illness and pneumonitis, but they can be hard to procure, and immune senescence complicates pathogenesis studies. We adapted the SARS-CoV (Urbani strain) by serial passage in the respiratory tract of young BALB/c mice. Fifteen passages resulted in a virus (MA15) that is lethal for mice following intranasal inoculation. Lethality is preceded by rapid and high titer viral replication in lungs, viremia, and dissemination of virus to extrapulmonary sites accompanied by lymphopenia, neutrophilia, and pathological changes in the lungs. Abundant viral antigen is extensively distributed in bronchial epithelial cells and alveolar pneumocytes, and necrotic cellular debris is present in airways and alveoli, with only mild and focal pneumonitis. These observations suggest that mice infected with MA15 die from an overwhelming viral infection with extensive, virally mediated destruction of pneumocytes and ciliated epithelial cells. The MA15 virus has six coding mutations associated with adaptation and increased virulence; when introduced into a recombinant SARS-CoV, these mutations result in a highly virulent and lethal virus (rMA15), duplicating the phenotype of the biologically derived MA15 virus. Intranasal inoculation with MA15 reproduces many aspects of disease seen in severe human cases of SARS. The availability of the MA15 virus will enhance the use of the mouse model for SARS because infection with MA15 causes morbidity, mortality, and pulmonary pathology. This virus will be of value as a stringent challenge in evaluation of the efficacy of vaccines and antivirals.

Figure 1. Schematic Diagram of SARS-CoV Genome Indicating Mutations Found in MA15 Virus

(A) The 29,727 nucleotide positive-sense RNA genome of SARS-CoV is depicted in this to-scale drawing with ORFs indicated by shaded boxes (dark gray, structural and non-structural proteins; light gray, accessory genes X1–X5 [37]; and straight lines, non-coding regions). Asterisks indicate the sites of the six nucleotide changes (compared with the published SARS-CoV (Urbani) sequence) resulting in six coding mutations found in the mouse-adapted SARS-CoV (MA15). (B) The six mutations found in MA15. ^aORF, open reading frame. ^bCDS, coding sequence, sequence of nucleotides that corresponds with the sequence of amino acids in a protein (location includes start and stop codon). ^cnsp, non-structural protein, cleavage product of ORF 1ab; Main^pro, main 3C-like protease; Hel, helicase. ^dRBM, receptor binding motif (amino acids 424–494).

Figure 2. Recombinant SARS-CoVs Demonstrate Normal Processing of vRNAs and Proteins

(A) Northern analysis. Intracellular RNA was isolated 10.5 h.p.i. from Vero E6 cells infected with indicated viruses or from mock-infected cells. RNA (0.1 μg) was treated with glyoxal, separated on 1% agarose gel, transferred to a BrightStar-Plus membrane, and probed with an N gene–specific biotinylated oligomer as described in Materials and Methods. (B) Western analysis. Cell lysates were separated on two 7.5% SDS-PAGE gels, transferred to polyvinylidene fluoride and probed with either mouse anti-S antisera (top panel) or probed first with a mouse anti-X1 antisera (sera raised to accessory protein X1 [37]; middle panel), and then stripped and probed again with a mouse anti-N antisera (bottom panel). Each primary antibody was followed by goat anti-mouse HRP-conjugated secondary antibody and visualized by enhanced chemiluminescence.

Figure 3. Virus Titers in Lungs of BALB/c Mice Inoculated with SARS-CoV or MA15 Virus

Data represents a compilation of two experiments. In each experiment, groups of four mice were inoculated intranasally with 50 μL of SARS-CoV (Urbani) (10^5.0 TCID₅₀/mouse, black bars) or MA15 virus at lethal (10^5.6 TCID₅₀/mouse, white bars) or sub-lethal (10^3.6 TCID₅₀/mouse, light gray bars) doses. Mice were sacrificed on indicated d.p.i. Mice receiving lethal doses of MA15 virus did not survive beyond day 4. Bars represent mean viral titers; error bars indicate standard error. Asterisks indicate significant differences (p < 0.05) compared with titers in mice receiving lethal doses of MA15 virus. Dotted line indicates lower limit of detection (10^1.5 TCID₅₀/g).

Figure 4. Histopathological Changes in Lungs from SARS-CoV (Urbani)–Infected and MA15-Infected Mice (Hematoxylin and Eosin Stain, Original Magnifications ×25)

Day 1 p.i. (A) SARS-CoV (Urbani)–infected mice: no significant inflammatory cell infiltrates. (B) MA15-infected mice: foci of perivascular, peribronchiolar, and interstitial inflammatory infiltrates comprised predominantly of mononuclear cells. Day 2 p.i. (C) Same as in (A). (D) MA15-infected mice: small, mononuclear inflammatory cell focus in the alveolar interstitium. Day 3 p.i. (E) SARS-CoV (Urbani)–infected mice: extensive and confluent interstitial pneumonitis. (F) MA15-infected mice: mild interstitial inflammation with diffuse pyknosis and karyorrhexis of alveolar pneumocytes. Day 4 p.i. (G) SARS-CoV (Urbani)–infected mice: small, discrete inflammatory cell infiltrates involving the alveolar interstitium. (H) MA15-infected mice: mild interstitial inflammation and necrotic intraalveolar debris. Mice were inoculated with 10^5.6 TCID₅₀ MA15 virus/mouse or 10^5.0 TCID₅₀ SARS-CoV (Urbani)/mouse.

Figure 5. Immunohistochemical Staining for SARS-CoV Antigen in Lungs of SARS-CoV(Urbani)–Infected or MA15-Infected Mice

Immunohistochemical staining is shown in red. Day 1 p.i. (A) SARS-CoV (Urbani)–infected mice: antigen present in occasional ciliated respiratory epithelial cells in bronchioles. (B) MA15-infected mice: diffuse and extensive staining of bronchiolar respiratory epithelium and alveolar pneumocytes. Day 2 p.i. (C) SARS-CoV (Urbani)–infected mice: staining of occasional bronchiolar epithelial cells and rare alveolar pneumocytes. (D) MA15-infected mice: staining predominantly in intact and detached cells in pulmonary alveoli. Day 3 p.i. (E) SARS-CoV (Urbani)–infected mice: occasional staining of alveolar pneumocytes and bronchiolar epithelium. (F) MA15-infected mice: extensive staining of abundant, intraalveolar, necrotic debris. Day 4 p.i. (G) SARS-CoV (Urbani)–infected mice: occasional focus of predominantly pneumocyte staining. (H) MA15-infected mice: extensive staining of abundant, intraalveolar, necrotic debris. Primary antibody, rabbit anti-SARS-CoV antibody; secondary antibody conjugated with alkaline phosphatase with naphthol fast-red and hematoxylin counterstain; original magnifications ×25. Mice were inoculated with 10^5.6 TCID₅₀ MA15 virus/mouse or 10^5.0 TCID₅₀ SARS-CoV (Urbani)/mouse.

Figure 6. Histopathology and Immunohistochemical Localization of SARS-CoV Antigens in the Lungs of Mice Infected with MA15 Virus

Abundant necrotic cellular debris (arrows) in alveoli (A) and a bronchiole lumen (B) of mice at days 2 and 3 p.i., respectively. Abundant SARS-CoV antigens (arrowheads) within alveolar pneumocytes (C) and in necrotic alveolar and bronchiolar cellular debris in mice at day 2 p.i. (D). (A and B) Hematoxylin and eosin stain; (C and D) primary antibody, rabbit anti-SARS-CoV antibody; secondary antibody conjugated with alkaline phosphatase with naphthol fast-red and hematoxylin counterstain; original magnifications ×100. Mice were inoculated with 10^5.6 TCID₅₀ MA15 virus/mouse.

Figure 7. Challenge of SARS-CoV– or Mock-Immunized Mice with the Lethal MA15 Virus

Groups of eight mice (8 wk old) were immunized intranasally with 50 μL of SARS-CoV (Urbani) (10⁵TCID₅₀/mouse) or L15 tissue culture media. Four weeks after immunization, mice were challenged intranasally with 50 μL MA15 virus (10^6.9 TCID₅₀/mouse), weighed daily, and observed twice daily for morbidity and mortality. Surviving mice that lost in excess of 20% initial body weight were euthanized. Symbols represent mean values for SARS-CoV–immunized mice (triangles) and mock-immunized mice (circles). Error bars indicate standard error.