Mouse-adapted MERS coronavirus causes lethal lung disease in human DPP4 knockin mice

Abstract

The Middle East respiratory syndrome (MERS) emerged in Saudi Arabia in 2012, caused by a zoonotically transmitted coronavirus (CoV). Over 1,900 cases have been reported to date, with ∼36% fatality rate. Lack of autopsies from MERS cases has hindered understanding of MERS-CoV pathogenesis. A small animal model that develops progressive pulmonary manifestations when infected with MERS-CoV would advance the field. As mice are restricted to infection at the level of DPP4, the MERS-CoV receptor, we generated mice with humanized exons 10-12 of the mouse Dpp4 locus. Upon inoculation with MERS-CoV, human DPP4 knockin (KI) mice supported virus replication in the lungs, but developed no illness. After 30 serial passages through the lungs of KI mice, a mouse-adapted virus emerged (MERSMA) that grew in lungs to over 100 times higher titers than the starting virus. A plaque-purified MERSMA clone caused weight loss and fatal infection. Virus antigen was observed in airway epithelia, pneumocytes, and macrophages. Pathologic findings included diffuse alveolar damage with pulmonary edema and hyaline membrane formation associated with accumulation of activated inflammatory monocyte-macrophages and neutrophils in the lungs. Relative to the parental MERS-CoV, MERSMA viruses contained 13-22 mutations, including several within the spike (S) glycoprotein gene. S-protein mutations sensitized viruses to entry-activating serine proteases and conferred more rapid entry kinetics. Recombinant MERSMA bearing mutant S proteins were more virulent than the parental virus in hDPP4 KI mice. The hDPP4 KI mouse and the MERSMA provide tools to investigate disease causes and develop new therapies.

Keywords: CD26; emerging pathogen; interferon; spike protein; virus pathogenesis.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

DPP4 immunostaining in uninfected hDPP4 KI mouse lung. (A) DPP4 was observed as mild to moderate cytoplasmic staining of airway epithelia. Positive staining was also noted in alveolar macrophages (arrow). (B) Mild to moderate macrophage immunostaining were commonly seen (arrows, Middle), as was weak staining of alveolar epithelia (Inset). (C) The pleural surface (arrows) had multifocal DPP4 immunostaining. Results shown are representative of findings from four hDPP4 KI mice. (Magnification: 400×.)

Fig. 2.

Outcomes in hDPP4 KI mice infected with EMC/Vero or MERSMA. Weight loss (A) and survival (B) in KI mice infected i.n. with 104 pfu EMC/Vero or passage 30 (P30) MERSMA. EMC/Vero-infected mice had 100% survival. Human DPP4 KI mice infected with P30 MERSMA lost significant weight and most died. Results summarize two replicate experiments, n = 10 (EMC/Vero), n = 11 (MERSMA), mixed male and female mice. (C–F) MERSMA clone 6.1.2 causes dose-dependent lethal lung disease in male and female hDPP4 KI mice. (C and D) Weight curve and survival results for male hDPP4 KI mice that received 5,000–50 pfu MERSMA clone 6.1.2 via i.n. inoculation. (For 5,000 pfu n = 8; 500 pfu n = 12; and 50 pfu n = 5). (E and F) Weight curve and survival results for female hDPP4 KI mice that received 5,000–50 pfu MERSMA clone 6.1.2 via i.n. inoculation. (For 5,000 pfu n = 8; 500 pfu n = 9; and 50 pfu n = 5). (A, C, and E) Data represent mean ± SE.

Fig. 3.

Tissue virus titers and viral genomic RNA distribution. Human DPP4 KI mice infected with 104 pfu i.n. inoculum of indicated MERS-CoV. (A and B) Lung and serum titers at indicated times (C) ORF1a quantitative RT-PCR performed on indicated tissues from EMC/Vero and MERSMA clone 6.1.2-infected mice at 1 and 3 d.p.i. Limit of detection (LOD) for qRT-PCR assay is ∼100 copies. Mean ± SE; n = 3. (D) Calu-3 cells were infected with indicated MERS-CoV at MOI 0.1 and virus titers at 1 d.p.i were determined by plaque assay. n = 6, representative of two replicate experiments. Statistical significance was assessed by Student’s t test. ****P < 0.0001.

Fig. 4.

Lung pathology and scoring at days 4–5 postinfection. (A) The uninfected group lacked lesions. EMC/Vero virus infection was characterized by multifocal mononuclear infiltrates (arrowheads), but mononuclear infiltrates were mild and uncommon in the clone 6.1.2-infected group. Rather, clone 6.1.2-infected lungs were characterized by multifocal hyaline membranes (A, arrows), edema, and necrotic debris that was consistent with diffuse alveolar disease: 40x (Top) and 200x (Bottom). (B and C) Further characterization of alveolar disease in EMC/Vero and clone 6.1.2 virus infection. Grossly (B, Top images), Evans blue dye (administered i.v. before killing) was not retained in EMC/Vero infection but was retained in clone 6.1.2 (white asterisks), indicative of vascular leakage. Histopathology (B, Bottom images) of EMC/Vero and clone 6.1.2 lungs. EMC/Vero-infected lung (Left) had perivascular to interstitial mononuclear infiltrates. Clone 6.1.2-infected lung exhibited edema (black asterisks, Bottom Middle, 400x), scattered cell death/debris (Inset and red arrow, Bottom Right), and/or hyaline membranes (black arrows, 600x). (C) Morphometry of lung tissues showed that clone 6.1.2 lungs had significantly reduced cellular infiltrate and increased hyaline membranes (P = 0.01 and P = 0.003, respectively, Mann–Whitney). n = 5–6 mice per condition.

Fig. 5.

MERS-CoV N-protein immunolocalization in uninfected KI mice or mice infected with EMC/Vero or MERSMA clone 6.1.2. No antigen staining was observed in uninfected hDPP4 KI mice (Left). At 3 d.p.i., mice infected with EMC/Vero had a patchy distribution of virus antigen (brown stain), including macrophage immunostaining along with uncommon epithelia of alveoli and airways (Middle). In contrast, clone 6.1.2-infected mice had widespread infection with staining of macrophages, alveolar epithelia, and scattered airway cells (Right). n = 3–4 mice per condition. [Magnification: 100x (Top) and 400x (Bottom.]

Fig. 6.

Accumulation of activated innate immune cells in the lungs of clone 6.1.2-infected hDPP4 KI mice. Lungs harvested from uninfected hDPP4 KI mice or hDPP4 KI mice challenged with 1 × 104 pfu of EMC/Vero or MERS MA clone 6.1.2 were analyzed for infiltrating immune cells on day 4 p.i. (A) FACS plots (Left) show percentages of leukocytes, inflammatory monocyte–macrophages, and neutrophils in the lungs of uninfected, EMC/Vero, and clone 6.1.2 groups. (B) Scatterplots (Right) represent percentages and total numbers of immune cells in the lungs of uninfected, EMC/Vero, and clone 6.1.2 groups. (C and D) Histograms and scatterplots show levels of activation markers, CD69 and CD80 on lung-derived IMMs (C), and neutrophils (D). (E) Bar graphs with scatterplots show total number of NK, T, and B cells in the lungs of uninfected, EMC/Vero, and clone 6.1.2 groups. Data were derived from two independent experiments with three to six mice per group per experiment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 7.

Adapted mutations in spike protein influence virus entry. (A) Linear depiction of MERS-CoV S1 (receptor binding) and S2 (fusion) domains. Amino acid changes in EMC/2012 and mouse-adapted MERS-CoV S proteins (MA1.0 and MA2.0) are indicated relative to the parent virus EMC/Vero. (B and C) Effects of E64d and camostat on MERS pseudovirus entry into LET1 cells. (D) Kinetics of MERS pseudovirus entry into LET1 cells. Error bars represent mean ± SE *P < 0.05, **P < 0.01, ***P < 0.001 compared with EMC/2012 S.

Fig. 8.

Influence of adapted spike protein mutations on in vivo virulence. (A and B) KI mice (age 12–13 wk, male) received 104 pfu of indicated recombinant MERS-CoV and were monitored daily for weight (A) and mortality (B) (n = 8 per group, representative of two replicate experiments). (C and D) KI mice (age 12–18 wk, male) received 105 pfu of indicated recombinant MERS-CoV and were monitored daily for weight (C) and mortality (D) (n = 10 per group, representative of two replicate experiments). (E) Lung virus titers at 1 d.p.i. determined by plaque assay in animals that received the 105 pfu inoculum (n = 6 per group, representative of two replicate experiments). (F) Calu-3 and Huh7 cells were infected with indicated recombinant viruses at MOI = 0.1. Progeny were collected at 1 d.p.i. and titered by plaque assay. (n = 6, representative of two replicate experiments). Error bars represent mean ± SE *P < 0.05, **P < 0.01, ***P < 0.001 compared with rMERS-CoV EMC/2012.