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

<div><p>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).</p><p>(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.</p></div

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

<div><p>(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 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030005#ppat-0030005-b037" target="_blank">37</a>]; 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).</p><p>(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).</p></div

Recombinant SARS-CoVs Demonstrate Normal Processing of vRNAs and Proteins

<div><p>(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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030005#s4" target="_blank">Materials and Methods</a>.</p><p>(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 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030005#ppat-0030005-b037" target="_blank">37</a>]; 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.</p></div

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

<p>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 (<i>p</i> < 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).</p

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

<p>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.</p

An 80% Plaque Reduction Neutralization Titers (PRNT₈₀) for VRP-S and VRP-S+N Hyperimmune Serum

<div><p>(A) Mice vaccinated young: icSARS-CoV (left) and icGDO3-S (right) PRNT₈₀ for VRP-S immune serum (experiment 2) collected at 5 wk post-boost (<i>n</i> = 5) and 53 wk post-boost (<i>n</i> = 8).</p> <p>(B) Mice vaccinated old: icSARS (left) and icGDO3-S (right) PRNT₈₀ values for VRP-S and VRP-S+N immune serum (experiment 4) at 12 and 29 wk post-boost (<i>n</i> = 6 for icSARS; <i>n</i> = 5 for icGDO3-S). The PRNT₈₀ values for individual animals are show as black circles, and the mean value is shown as a solid bar. The limits of detection (1:1,600 upper and 1:100 lower) are represented by horizontal dotted lines.</p></div

VRP-S Induces Short- and Long-Term Protection against icSARS-CoV Challenge

<div><p>icSARS titers are expressed as the log₁₀ plaque-forming units per gram (pfu/g) of lung. Tissues were homogenized in PBS to form a 20% suspension and titered on Vero monolayers. The titers for individual mice are shown as a filled circle, and the mean titer for the group is represented by a solid bar. Limit of detection (lod) is 2.4 log₁₀ pfu/g.</p> <p>(A) Lung titers of16-wk-old BALB/c mice harvested 2 d after being i.n. infected with 10⁵ PFU of icSARS-CoV (<i>n</i> = 6).</p> <p>(B) Lung titers of BALB/c mice vaccinated and boosted with 10⁶ infectious units (IU) of VRP expressing the influenza HA (VRP-HA), SARS-S glycoprotein (VRP-S), SARS-N protein (VRP-N), or a combination of VRP-S and VRP-N (VRP-S+N). Mice (<i>n</i> = 7 VRP-HA, <i>n</i> = 8 for other groups) were vaccinated at 5 wk of age, boosted 5 wk later, then i.n. challenged with 10⁵ pfu of icSARS-CoV 54-wk post-boost. Lungs were harvested 4 d later and titered.</p> <p>(C) Plaque assay results were confirmed by in situ hybridization to sectioned lungs of five mice from each vaccinated group with a radiolabeled riboprobe complementary to the SARS CoV N gene. In senescent mice challenged with the icSARS-CoV, representative lung sections from VRP-HA– (unpublished data) and VRP-N–vaccinated (a) animals exhibited extensive in situ signal (black arrows), whereas only one of five sections from VRP-S–vaccinated (b) and zero of five sections from VRP-S+N–vaccinated (c) mice exhibited SARS-CoV–specific signal above background levels.</p></div

Identifying Eosinophils among Inflammatory Infiltrates

<p>The 400× magnification comparing eosinophil infiltration within the lung sections of VRP-HA–vaccinated (A), (C), (E), and (G) and VRP-N–vaccinated (B), (D), (F), and (H) mice (<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030525#pmed-0030525-t001" target="_blank">Table 1</a>, Experiment 5). At day 2 post-infection (A) and (B), eosinophils are rarely evident in the lungs of either VRP-HA (A) or VRP-N (B) mice. Day 4 post-infection (C) and (D), extensive eosinophils (yellow arrows) are present within the lungs of VRP-N–vaccinated mice. Widespread eosinophils are seen at day 7 post-challenge in VRP-N–vaccinated (F), but not VRP-HA–vaccinated (E) mice. By day 14 (G) and (H), eosinophils are rarely found among inflammatory cells of VRP-N–vaccinated mice. An identical experiment in old animals was performed simultaneously (<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030525#pmed-0030525-t001" target="_blank">Table 1</a>, experiment 6), showed results indistinguishable from those of young mice (unpublished data). All tissues were stained with hematoxylin and eosin.</p

Synthetic Reconstruction of icGD03-S

<div><p>(A) Unrooted phylogenetic gene tree of 35 SARS isolates ranging from early, middle, and late phases of the 2002–2003 epidemic to 2003–2004 animal isolates. Branch confidence values are shown as posterior probabilities. The three human isolates that fall within the cluster otherwise isolated from animals (shown in boxes), GZ0402, GD03, and GZ0401, may represent infections in which a human acquired the virus from a Himalayan palm civet.</p> <p>(B) The GDO3-S glycoprotein. Amino acid changes unique to the GDO3-S with the GDO3-S amino acid listed on the left and the corresponding Urbani to the right. The GDO3-S amino acid changes are shown in relation to the S1 and S2 subunits, the receptor binding domain (RBD), heptad repeats one (HR1) and two (HR2), the transmembrane domain (TM), and known neutralizing epitopes. Two mutations that arose during tissue culture passage of the chimeric icGDO3-S are shown in red.</p> <p>(C) Growth curves of the Urbani strain of SARS-CoV (diamond, solid line), the recombinant Urbani icSARS (squares, dashed line), and the recombinant chimeric virus icGDO3-S (triangles, dotted line) in human airway epithelial cells.</p> <p>(D) Comparing growth of icSARS-CoV to icGDO3-S in the lungs of mice. Six-week-old female BALB/C mice were infected with icSARS-CoV or icGDO3-S (<i>n</i> = 5 per group). The individual titer of each mouse is represented by a filled circle, and the mean titer of the group is represented as a solid bar.</p></div

Pathogenic Findings Following Homologous Challenge

<p>Light photomicrographs of representative histologic lung sections (<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030525#pmed-0030525-t001" target="_blank">Table 1</a>, experiment 2) taken from an untreated control mouse (A), a VRP-N–vaccinated mouse (B) and (D), a VRP-HA–vaccinated mouse (C), a VRP-S–vaccinated mouse (E), and a VRP-S– and VRP-N–treated mouse (F). No histopathology was evident in (A). A marked mixed inflammatory infiltrate composed mainly of mononuclear leukocytes (lymphocytes and plasma cells) and widely scattered eosinophils are evident in the perivascular and peribronchiolar interstitium (asterisk) in (B). Similar inflammatory cells are also present in bronchiolar (br) airways and alveolar airspaces along with enlarged and vacuolated alveolar macrophages (arrows). The box in (B) denotes the site of the light photomicrograph (D) that was taken at a higher magnification to better illustrate the lymphoplasmacytic inflammatory cell infiltrate with lesser numbers of eosinophils (arrows). Similar, but slightly less severe, perivascular inflammatory infiltrates (asterisk) are also present in (F), but without accompanying alveolitis. Minimal lymphoplasmacytic cell accumulations around the pulmonary arteriole (a) are evident in (C) and (E). All tissues were stained with hematoxylin and eosin. Bars denote the scale of the magnification. a, pulmonary arteriole; ap, alveolar parenchyma; br, bronchiolar lumen; e, surface epithelium of the bronchiole.</p