Generation of a transgenic mouse model of Middle East respiratory syndrome coronavirus infection and disease

Abstract

The emergence of Middle East respiratory syndrome-coronavirus (MERS-CoV) in the Middle East since 2012 has caused more than 900 human infections with ∼40% mortality to date. Animal models are needed for studying pathogenesis and for development of preventive and therapeutic agents against MERS-CoV infection. Nonhuman primates (rhesus macaques and marmosets) are expensive models of limited availability. Although a mouse lung infection model has been described using adenovirus vectors expressing human CD26/dipeptidyl peptidase 4 (DPP4), it is believed that a transgenic mouse model is needed for MERS-CoV research. We have developed this transgenic mouse model as indicated in this study. We show that transgenic mice globally expressing hCD26/DPP4 were fully permissive to MERS-CoV infection, resulting in relentless weight loss and death within days postinfection. High infectious virus titers were recovered primarily from the lungs and brains of mice at 2 and 4 days postinfection, respectively, whereas viral RNAs were also detected in the heart, spleen, and intestine, indicating a disseminating viral infection. Infected Tg(+) mice developed a progressive pneumonia, characterized by extensive inflammatory infiltration. In contrast, an inconsistent mild perivascular cuffing was the only pathological change associated with the infected brains. Moreover, infected Tg(+) mice were able to activate genes encoding for many antiviral and inflammatory mediators within the lungs and brains, coinciding with the high levels of viral replication. This new and unique transgenic mouse model will be useful for furthering knowledge of MERS pathogenesis and for the development of vaccine and treatments against MERS-CoV infection.

Importance: Small and economical animal models are required for the controlled and extensive studies needed for elucidating pathogenesis and development of vaccines and antivirals against MERS. Mice are the most desirable small-animal species for this purpose because of availability and the existence of a thorough knowledge base, particularly of genetics and immunology. The standard small animals, mice, hamsters, and ferrets, all lack the functional MERS-CoV receptor and are not susceptible to infection. So, initial studies were done with nonhuman primates, expensive models of limited availability. A mouse lung infection model was described where a mouse adenovirus was used to transfect lung cells for receptor expression. Nevertheless, all generally agree that a transgenic mouse model expressing the DPP4 receptor is needed for MERS-CoV research. We have developed this transgenic mouse model as indicated in this study. This new and unique transgenic mouse model will be useful for furthering MERS research.

FIG 1

Transfection with the hCD26 transgene construct, pCAGGS.hCD26, effectively converts nonsusceptible mouse fibroblastic 17CL-1 cells to become fully supportive of productive MERS-CoV infection. (A) Schematic diagram of hCD26 expressing vector cassette, designated pCAGGS.hCD26. The hCD26 gene was cloned in this vector by restriction digestion with EcoRI at the multiple cloning site (MCS) which is driving the expression in the presence of chicken β-actin promoter. Introns and polyadenylation signals were included in the expressing cassette to enhance the transcription and increase the mRNA stability of transgenes. These elements were cloned into the plasmid such that the transgene could be expressed in one piece from the plasmid backbone by digestion with restriction enzymes (38, 39). Confluent parental and 17CL-1 cells stably transfected with the hCD26 transgene construct were analyzed for the expression of hCD26 with a goat anti-hCD26 antibody using indirect immunofluorescent staining (B) and Western blot analysis (C). The expression of hCD26 antigen was detected exclusively in 17CL-1/hCD26 cells as the green fluorescent protein with an expected size of 110 kDa. (D to F) To assess the permissiveness of 17CL-1/hCD26 cells to MERS-CoV infection, confluent 17CL-1 and 17CL-1/hCD26 cells, grown in 12-well plates, were infected with either MERS-CoV/EMC-2012 or rMERS-CoV/RFP at an MOI of 1 and monitored for CPE for 2 days after infection (D and E), and yields of infectious progeny virus were titrated at 1 and 2 dpi (F; the dashed line indicates the detection limit). The data shown are representative of at least two independently conducted experiments. The infectious virus titer (log₁₀ TCID₅₀/ml) is expressed as the mean ± the standard error of triplicate samples. ***, P ≤ 0.001 (determined using the Student t test, comparing 17CL-1 and 17CL-1/hCD26 cells).

FIG 2

Expression of hCD26 in transgene-positive (Tg⁺) mice. (A) Genomic DNA extracted from tail biopsy specimens of hCD26 transgenic founder mice of lines 52, 62, and 72 were digested by BamHI and subjected to Southern blot analyses to determine the relative intensities of the integrated hCD26 transgene, as described in Materials and Methods. The relative intensities of hCD26 among these three Tg⁺ lineages are presented as ratios of the signals probed by a SalI-NcoI fragment (CMV enhancer) and a 0.7-kb BglII fragment (DPP4 3′UTR, isolated from pCAGGS.hCD26, and Gsc2 5′) and used to normalize the amount of DNA. (B) Tg⁺ mice derived from lines 52 and 72 were euthanized, and their various tissue specimens were harvested to extract total RNA to assess the relative tissue abundances of hCD26 mRNA expression by qRT-PCR, as described in Materials and Methods. (C) hCD26 Tg⁺ mice of line 52 were euthanized, and the homogenates and paraffin-embedded sections of various tissues were prepared to assess the expression of hCD26 protein by Western blotting with the anti-hCD26 antibody described in Fig. 1. A varying intensity of hCD26 protein was detected in all of the tissues analyzed. (D) Paraffin-embedded tissues derived from Tg⁺ and Tg⁻ mice were subjected to IHC staining with an anti-hCD26 antibody to localize hCD26 expression. In Tg⁺ mice, the expression of hCD26 in the lungs was readily detectable in type I and type II alveolar pneumocytes (arrow), endothelial cells (arrow), and neurons (arrowhead) in the brain, muscle cells (arrow) in the heart, endothelial cells (arrow) and the surface of hepatocytes (arrowhead) in the liver, endothelial cells (arrow) in the kidneys, and epithelial lining (arrowhead) and the underneath muscularis (arrow) in the intestines. No hCD26 expression was evident in the tissues of Tg⁻ mice.

FIG 3

Transgenic mice expressing hCD26 are permissive to MERS-CoV infection, leading to morbidity and mortality. Both hCD26 Tg⁺ and Tg⁻ mice, nine animals each, were challenged i.n. with 10⁶ TCID₅₀ of MERS-CoV in 80 μl. Infected mice were monitored daily for their weight gain or loss (A), other signs of clinical illness, and mortality (B). Two animals were euthanized at 2 and 4 dpi to determine the viral loads in various tissues harvested by quantifying infectious virus isolated (C) and viral RNA targeting upstream E gene by qRT-PCR (D). In addition, paraffin-embedded lung (E and F) and brain (G and H) tussues were stained for MERS-CoV antigen with a specific anti-MERS-CoV antibody raised in rabbits, as described in Materials and Methods. (A) Persistent weight loss (up to 30%) in infected Tg⁺, but not Tg⁻, mice. (B) Cumulative survival rate of infected mice. Tg⁺ mice succumbed to infection with 100% mortality at 6 dpi. (C) Infectious virus titers, expressed as log₁₀ TCID₅₀ per g of tissue, in the lungs and brain. (D) Copy numbers of viral E gene in the indicated tissues of Tg⁺ mice, relative to those derived from Tg⁻ littermates. (E and F) Viral antigen (red) was readily detected in alveolar pneumocytes of Tg⁺ (E), but not Tg⁻ (F), mice at 2 dpi. (G and H) Viral antigen (red) was also detected in neurons (green arrow), microglia (arrowhead), astrocytes (black arrow), and astrocyte processes (star) in the brains of Tg⁺ (G), but not Tg⁻ (H), mice at 4 dpi.

FIG 4

Histopathological changes in the lungs and brains of hCD26 Tg⁺ mice challenged with MERS-CoV. Tg⁺ and Tg⁻ mice were euthanized on day 2 and 4 after challenge with MERS-CoV for assessing the pathology of the lungs and brain. (A) More extensive gross lesions of the lungs were detected in Tg⁺ mice than in Tg⁻ mice, starting at day 2 after challenge. (B) Paraffin-embedded sections of lung and brain specimens were stained with H&E. Compared to a few foci of perivascular infiltration in the lungs of Tg⁻ mice, a more intense and widespread bronchiolitis and alveolitis was observed in the lungs of Tg⁺ mice, starting at day 2 postchallenge. The major cell types of the inflammatory infiltrates were lymphocytes and monocytes, with a few scattered neutrophils.

FIG 5

MERS-CoV infection induces profound acute innate inflammatory responses within the lungs and brain of hCD26 Tg⁺ mice. Total RNAs were extracted from the lungs and brains of Tg⁺ and Tg⁻ mice at days 2 and 4 after infection with MERS-CoV and processed for profiling innate inflammatory responses by qRT-PCR, targeting a panel of 14 genes encoding antiviral and proinflammatory cytokines and chemokines. The relative abundance of mRNA transcripts of each gene was calculated according to the comparative ΔΔC_T method. The results are presented as the fold changes in gene expression of infected Tg⁺ mice compared to that of infected Tg⁻ mice. The data displayed represent the means ± the standard errors of two animals, with duplicate samples of each.