Isolation and characterization of a pangolin-borne HKU4-related coronavirus that potentially infects human-DPP4-transgenic mice

Abstract

We recently detected a HKU4-related coronavirus in subgenus Merbecovirus (named pangolin-CoV-HKU4-P251T) from a Malayan pangolin¹. Here we report isolation and characterization of pangolin-CoV-HKU4-P251T, the genome sequence of which is closest to that of a coronavirus from the greater bamboo bat (Tylonycteris robustula) in Yunnan Province, China, with a 94.3% nucleotide identity. Pangolin-CoV-HKU4-P251T is able to infect human cell lines, and replicates more efficiently in cells that express human-dipeptidyl-peptidase-4 (hDPP4)-expressing and pangolin-DPP4-expressing cells than in bat-DPP4-expressing cells. After intranasal inoculation with pangolin-CoV-HKU4-P251, hDPP4-transgenic female mice are likely infected, showing persistent viral RNA copy numbers in the lungs. Progressive interstitial pneumonia developed in the infected mice, characterized by the accumulation of macrophages, and increase of antiviral cytokines, proinflammatory cytokines, and chemokines in lung tissues. These findings suggest that the pangolin-borne HKU4-related coronavirus has a potential for emerging as a human pathogen by using hDPP4.

Fig. 1. The isolation and characteristics of pangolin-CoV-HKU4-P251T.

a The viral load of pangolin-CoV-HKU4-P251T in series passages in Vero 81 cell cultures. The viral load are expressed as copies per microliter (µL). b Negative stain electron microscopy verified the extracellular virus particle of pangolin-CoV-HKU4-P251T. Original magnification ×80 K. c Fluorescence in situ hybridization locating ORF1ab gene of pangolin-CoV-HKU4-P251T at 48 HPI. Nuclei, DAPI (blue); ORF1ab probe, Quasar 570 (red). Representative microscopy fields are shown. Original magnification ×200. Each imaging experiment was performed at least three times independently with similar results, and the representative microscopy fields are shown (b and c). d Genetic variation analysis of pangolin-CoV-HKU4-P251T during in vitro passage. The parental pangolin-CoV-HKU4-P251T strain (GenBank accession No. OM009282) was used as the reference sequence. Source data are provided as a Source Data file.

Fig. 2. Efficient replication of pangolin-CoV-HKU4-P251T in human cell lines.

a Kinetics of replication of pangolin-CoV-HKU4-P251T in human Huh7, Caco-2, Calu-3, and BEAS-2B cell lines. Viral RNA levels of virus in cell culture supernatant were detected at 0, 12, 24, 48, and 72 HPI, with three independent biological replicates per time point and three technical replicates per sample. The viral RNA levels were normalized relative to initial phase of infection (0 HPI). Data are presented as mean ± SD (shown as error bars). ANOVA was used for multiple comparisons. b Fluorescence in situ hybridization locating ORF1ab gene of Pangolin-CoV-HKU4-P251T in each cell lines at 48 HPI. Nuclei, DAPI (blue); ORF1ab probe, Quasar 570 (red). Representative microscopy fields are shown. Original magnification ×200. See also Supplementary Fig. 2. c Western blotting analyses of expressions of hDPP4 and hACE2 in various cell lines. Α-Tubulin was used as the loading control. d Cytopathic effect (CPE) of virus-infected Huh7 cells at 5 DPI. Original magnification×100. The uninfected Huh7 cells cultivated in parallel are used as controls. Each imaging experiment was independently performed at least three times with similar results, and representative images are shown (b–d). e Viral titers of infected Huh7 cells at different time points post-infection. n = 3 biologically independent experiments per time point. Data are presented as mean ± SD (shown as error bars). ANOVA was used for multiple comparisons. f The growth curve of passages 1 and 15 viruses in Huh7 cells. Cells were infected with the virus at an MOI of 0.01. At the specified time intervals, the supernatants were collected and the viral titers were measured as instructed. n = 3 biologically independent experiments per time point. Data are displayed as a line representing the mean, with individual data points shown as dots. Source data are provided as a Source Data file.

Fig. 3. DPP4 usage of pangolin-CoV-HKU4-P251T In Vitro.

Western blotting assay showing the expression of DPP4 (a) and ACE2 (c) in transient transfected cells. GAPDH was used as the loading control. Pangolin-CoV-HKU4-P251T growth in HeLa cells overexpression DPP4 (b) and ACE2 (d). The HeLa-WT cells were used as a negative control. Fold increases of viral copies are determined by comparing to the viral copies in HeLa-WT cells. Data are presented as mean ± SD (shown as error bars) of three independent biological replicates per time point and three technical replicates per sample. ANOVA was used for multiple comparisons. e, f Indirect immunofluorescence staining. Representative images show the expression of receptor protein (green) and viral nucleocapsid protein (red) in different cell lines at 48 HPI. Nuclei, DAPI (blue). Original magnification ×200. Each imaging experiment was performed independently at least three times with similar results, and representative images of the results are shown (a, c, e, and f). Source data are provided as a Source Data file.

Fig. 4. Pangolin-CoV-HKU4-P251T infection in the lungs of hDPP4-transgenic mice.

a The copy number of viral RNA in lungs of pangolin-CoV-HKU4-P251T-infected hDPP4 (orange) and WT (blue) mice was determined using qRT-PCR. n = 3 biologically independent animals per genotype for each time point. Data are presented as mean ± SD with scatter plot at each time point. Dashed lines denote the detection limit. Whenever the sample measurement was below the detection limit, the result was assigned a value equal to the minimum detection limit to facilitate statistical analysis of the data. ANOVA was used for multiple comparisons. b Relative sgmRNA quantification in lungs of virus-infected hDPP4 (orange) and WT (blue) mice. n = 3 biologically independent animals per genotype for each time point. Data are presented as mean ± SD with scatter plot at each time point. ANOVA was used for multiple comparisons. c The expression of viral nucleocapsid protein in lungs of infected hDPP4-mice. Representative IHC staining images of viral nucleocapsid protein expression in lungs of infected hDPP4-mice at 3, 6, and 12 DPI. The hDPP4-mice inoculated with cell medium are used as control. Images are representative of three experimental animals. Original magnification ×200 (upper panel). Red frames indicate regions shown in high magnification (lower panel, ×400). See also Supplementary Fig. 3 and 4a. Source data are provided as a Source Data file.

Fig. 5. Pathogenicity of virus-infected hDPP4-transgenic mice.

a Percentage of body weight changes of infected hDPP4-mice and WT-mice. n = 17 biologically independent animals per genotype. Data are reported as mean ± SD (shown as error bars). The solid dots and lines indicate the weight changes of hDPP4-mice. The hollow dots and dashed lines indicate the weight changes of WT-mice. Orange, live virus-infected mice; blue, heat-inactivated viruses-infected mice; dark gray, cell culture supernatant-infected mice. ANOVA was used for multiple comparisons. b Pathological features of virus-infected hDPP4-mice. H&E-stained lung sections at 3, 6, and 12 DPI are displayed. The hDPP4-mice inoculated with cell medium are used as control. Images are representative of three experimental animals. Original magnification ×200. See also Supplementary Fig. 4b. c Immunohistochemical analysis of lung tissue stained with MAC2 antibody for macrophage detection. The hDPP4-mice inoculated with cell medium are used as control. Images are representative of three experimental animals. Original magnification ×400. See also Supplementary Fig. 4c. d Cytokine response in the lung of hDPP4 (orange) and WT (blue) mice following virus infection. mRNA levels were monitored by qPCR at 1, 3, 6, and 12 DPI. The relative expression of mRNA at 3, 6, and 12 DPI was measured by comparative 2^−∆∆CT method related to 1 DPI and the mean ± SD (shown as error bars) of fold change of three independent biological replicates for each gene at each time point are presented. Student’s T-test (two-tailed) was used for comparisons of hDPP4- and WT-mice. Source data are provided as a Source Data file.