Lethal infection of K18-hACE2 mice infected with severe acute respiratory syndrome coronavirus - PubMed (original) (raw)

doi: 10.1128/JVI.02012-06. Epub 2006 Nov 1.

Lecia Pewe, Christine Wohlford-Lenane, Melissa Hickey, Lori Manzel, Lei Shi, Jason Netland, Hong Peng Jia, Carmen Halabi, Curt D Sigmund, David K Meyerholz, Patricia Kirby, Dwight C Look, Stanley Perlman

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Lethal infection of K18-hACE2 mice infected with severe acute respiratory syndrome coronavirus

Paul B McCray Jr et al. J Virol. 2007 Jan.

Abstract

The severe acute respiratory syndrome (SARS), caused by a novel coronavirus (SARS-CoV), resulted in substantial morbidity, mortality, and economic losses during the 2003 epidemic. While SARS-CoV infection has not recurred to a significant extent since 2003, it still remains a potential threat. Understanding of SARS and development of therapeutic approaches have been hampered by the absence of an animal model that mimics the human disease and is reproducible. Here we show that transgenic mice that express the SARS-CoV receptor (human angiotensin-converting enzyme 2 [hACE2]) in airway and other epithelia develop a rapidly lethal infection after intranasal inoculation with a human strain of the virus. Infection begins in airway epithelia, with subsequent alveolar involvement and extrapulmonary virus spread to the brain. Infection results in macrophage and lymphocyte infiltration in the lungs and upregulation of proinflammatory cytokines and chemokines in both the lung and the brain. This model of lethal infection with SARS-CoV should be useful for studies of pathogenesis and for the development of antiviral therapies.

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Figures

FIG. 1.

FIG. 1.

Generation and characterization of K18-hACE2 mice. (A) The hACE2 coding sequence (CDS) was cloned into a construct containing 5′ and 3′ genomic regions of the human K18 gene, which had previously been shown to be necessary for driving high-level epithelial-cell-specific expression. The K18 5′ genomic region consists of a 2.5-kb upstream genomic sequence, the promoter, and the first intron of the human K18 gene, while the K18 3′ region consists of exon 6, intron 6, exon 7, and ∼300 bp of the 3′ UTR of the human K18 gene, including the K18 poly(A) signal. Immediately upstream of the hACE2 start codon is a translational enhancer (TE) sequence from alfalfa mosaic virus. (B) hACE2 cDNA copy numbers in three transgenic founder lines determined by quantitative PCR, as described in Materials and Methods.

FIG. 2.

FIG. 2.

hACE2 expression in K18-hACE2 mice. (A) hACE2 transgene mRNA expression levels in the indicated mouse tissues. Quantitative RT-PCR was used to determine the relative abundances of the hACE2 transgene in the tissues given along the x axis, as described in Materials and Methods. Results are means ± standard errors for 3 to 6 mice per group. (B) hACE2 transgene mRNA expression in brains of K18-hACE2 mice. Results are means ± standard errors for 3 to 6 mice per group. Note the change in scale from panel A. (C) Expression of mouse ACE2 mRNA in lungs and brains of non-Tg and K18-hACE2 transgenic mice as determined by quantitative RT-PCR. Results are means ± standard errors for 3 to 6 mice per group.

FIG. 3.

FIG. 3.

SARS-CoV causes lethal disease in K18-hACE2 mice. (A and B) K18-hACE2 mice (lines 1 [n = 15], 2 [n = 11], and 3 [n = 15]) and 15 non-Tg mice were infected intranasally with 2.3 × 104 PFU of SARS-CoV and were monitored daily for mortality (A) and weight (B). (C and D) Tissues were harvested from infected mice and assayed for infectious virus as described in Materials and Methods. Virus was detected only in the brains and lungs of K18-hACE2 mice and only in the lungs of non-Tg mice. Tissues from 3 to 6 mice were analyzed at each time point. Significantly more virus was detected in line 2 lungs at day 2 p.i. than in the lungs of non-Tg mice (P < 0.02). More virus was detected in line 3 lungs at day 4 p.i. (P < 0.0004), but not at day 2 p.i., than in the lungs of non-Tg mice.

FIG. 4.

FIG. 4.

Quantitative RT-PCR for the N gene of SARS-CoV. RNA was prepared from the lungs and brains of K18-hACE2 (line 3) and non-Tg mice at days 2 and 4 p.i. RNA levels were detected by quantitative RT-PCR as described in Materials and Methods. Viral RNA levels parallel levels of infectious virus (Fig. 1). RNAs from six mice were analyzed in all groups, except that three brains from each group were analyzed at day 2. Significantly more viral RNA was detected in K18-hACE2 lungs than in non-Tg lungs at days 2 and 4. For K18-hACE2 lungs, significantly more viral RNA was detected at 2 days p.i. than at 4 days p.i. (P < 0.005).

FIG. 5.

FIG. 5.

Pulmonary disease in SARS-CoV-infected K18-hACE2 and non-Tg mice. (A through J and L) K18-hACE2 and non-Tg mice were either left uninfected (A and B) or infected with 2.3 × 104 PFU of SARS-CoV. Lungs were fixed in zinc formalin and stained with hematoxylin and eosin. Non-Tg mice showed mild perivascular and peribronchiolar inflammation in response to SARS-CoV 2 (C) and 4 (I) days following infection. K18-hACE2 mice demonstrated more-extensive disease 2 days following infection, characterized by epithelial sloughing (D, arrowheads) and more-extensive areas of mixed inflammatory cell infiltrates within and around airways, blood vessels, and the alveolar parenchyma. At day 2 p.i., viral antigen was localized to conducting airway epithelia in non-Tg (E) and K18-hACE2 (F) mice. Cells recovered from BAL specimens of infected K18-hACE2 mice (H) included macrophages with more vacuoles, consistent with activation, as well as enhanced neutrophilia and lymphocytosis compared to non-Tg mice (G). By 4 days p.i., inflammation in infected non-Tg lungs was resolving (I), while perivascular and peribronchiolar infiltrates and hemorrhage (arrowhead) were detected in K18-hACE2 mice (J). In some animals, bronchioles were completely occluded by neutrophils with marked intra-alveolar edema and without vasculitis (L), consistent with aspiration. BAL specimens were obtained from uninfected and infected K18-hACE2 and non-Tg mice and results pooled for 3 and 4 days p.i. Bars, 50 μm. (K) BAL analysis. Means (standard errors) are shown. K18-hACE2 Tg mice exhibited increased numbers of lymphocytic and neutrophilic cells in BAL specimens compared to non-Tg mice. n ≥ 6 for all conditions except for naïve non-Tg mice (n = 3). *, P < 0.05 for comparison to naïve mice. Mac, macrophages; L, lymphocytes; PMN, neutrophils.

FIG. 6.

FIG. 6.

SARS-CoV infects the brains of K18_-hACE2_ but not non-Tg mice. Brains were harvested from infected K18-hACE2 (A to C) and non-Tg (D and E) mice and stained with hematoxylin and eosin (B and D) or for virus antigen (A, C, and E). (A) Virus (brown) is detected in large numbers of cells in the cerebrum (C), thalamus, and brainstem but not in the olfactory bulb (OB) or cerebellum (Ce). Brainstem (B) and cerebellum (Ce) tissues are shown in panels B to E. (B and D) Little inflammation is present in the brains of infected non-Tg or K18-hACE2 mice. (C) Extensive infection of neurons is detected in the brainstem but not in the adjacent cerebellum. (E) No antigen labeling is detected in the brains of non-Tg mice. Bar, 100 μm.

FIG. 7.

FIG. 7.

Detection of proinflammatory cytokine and chemokine mRNAs in the lungs and brains of infected K18-hACE2 and non-Tg mice. Infected K18-hACE2 (line 3) and non-Tg mice were sacrificed at day 2 p.i. (6 mice each) (A) and day 4 p.i. (6 mice each) (B and C). RNAs were prepared from lungs (A and B) and brains (C) and assayed for cytokine and chemokine mRNA levels by using an RNase protection assay as described in Materials and Methods. Data are shown as levels of RNA normalized to the level of a housekeeping gene (L32). (A and B) There were significant differences (P < 0.05) in pulmonary mRNA levels of CCL7, CCL12, CXCL10, and IL-12p35 between K18-hACE2 and non-Tg mice at day 2 (A). Differences in levels of tumor necrosis factor alpha and IL-6 were nearly significant (P < 0.06). At day 4 p.i., there was a significant difference in IL-1β levels between K18-hACE2 and non-Tg mice (B). There was a statistically significant decrease (P < 0.05) in the levels of all cytokines and chemokines when infected K18-hACE2 mice at days 2 and 4 p.i. were compared, except for CXCL10 (P = 0.06) and IL-1β (P = 0.47). IL-2, IL-4, IL-10, IL-12p40, IFN-β, IFN-α, CCL3, and CXCL2 (MIP-2) were not detected in lungs. (C) Levels of all cytokine and chemokine mRNAs in infected K18-hACE2 and non-Tg brains were indistinguishable from those of naïve brains at day 2 p.i. (data not shown). By day 4 p.i., all cytokine and chemokine mRNA levels were statistically higher in K18-hACE2 mice than in non-Tg mice (P < 0.02). IL-2, IL-4, IL-10, IFN-α, CCL3, and CXCL2 were not detected in brains. Stippled bars, naive mice; open bars, non-Tg mice; solid bars, K18-hACE2 mice.

FIG. 8.

FIG. 8.

Treatment with an anti-SARS-CoV neutralizing antibody protects K18-hACE2 mice against clinical disease. K18-hACE2 mice (line 2) received 25 mg of MAb 201 (9 mice) or a control antibody (7 mice)/kg 1 day prior to infection with 2.3 × 104 PFU of SARS-CoV. Mice were monitored for survival and weight loss. All infected K18-hACE2 mice that received MAb 201 survived and exhibited no weight loss (data not shown).

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