A Mouse-Adapted Model of HCoV-OC43 and Its Usage to the Evaluation of Antiviral Drugs

Figure 1. The expression of viral n gene in the brain and lungs of a series generations of mice and mutation sites between wild and adapted strains. (A) The expression of viral n gene in the brain and lung was detected at 4 dpi (n = 3–5). The data are presented as the log10 of the expression of viral n gene (copies/g), the mean and the standard error of the mean (SEM). *p < 0.05; **p < 0.01; and ***p < 0.001; vs. P1. (B) A schematic representation of the HCoV-OC43-1558 HE and S proteins (drawn to scale) and the five amino acid substitutions. The hemagglutinin-esterase protein is colored in blue, the S1 subunit and S2 subunit of S protein are colored in pink and orange, respectively, and the S1 domains A, B, C, and D are colored in green, purple, yellow, and gray, respectively. The substitution of Y250H, N259D, and D264K are located in domain A of the S1 subunit, while A394G and K494I are located in domain B. (C) Schematic diagram of HCoV-OC43 genome indicating mutations found in P9 virus. (Top) The 30,746 nucleotide RNA genome of HCoV-OC43 is shown in this to scale drawing with ORFs indicated by gray boxes. Black asterisks indicate nucleotide mutations which did not result in coding…

Figure 2. Body weight changes and survival rates in suckling mice infected with wild and adapted strains. After inoculation with a wild-type strain virus (WT) or an adapted-strain virus (P9), body weight changes (A), and survival rates (B) of suckling mice within 15 days. The data are presented as the mean and SEM (n = 10).

Figure 3. Virus expression and organ indices in various organs of suckling mice. The proliferation kinetics of these viruses in the brain and lungs was analyzed (n = 3); (A) Virus n gene’s copy number (B) and organ indices (C) in various organs of mice infected with wild-type strain viruses (WT) and adapted-strain viruses (P9) were detected at 4 dpi (n = 4), respectively. The viral replication levels (D) and organ indices (E) in WT-infected suckling mice at 8 dpi and P9-infected at 5 dpi (n = 3–4) were calculated. The data for viral n gene (copies/g) are presented as log10, mean ± SEM, and dashed lines indicate the detection limit.

Figure 4. Virus N protein expression and localization in organs of suckling mice. Viral nucleocapsid protein expression and localization in brain (400×) and lung (400×) tissues were examined immunohistochemically with DAB staining at the indicated times (n = 3–5). The OC43 N protein expression level was calculated and indicated as H-Scores. Positive expression is shown in tan. Blue arrows, bronchus. #p < 0.05, ##p < 0.01, and ###p < 0.001, vs. WT-8 dpi group; **p < 0.01, vs. WT-4 dpi group.

Figure 5. Pathological changes and expression of pro-inflammatory factors in brain tissues induced by virus infection. (A) HE staining of brain tissue at 3, 4, and 8 dpi after virus WT or P9 infection, 400×, n = 3–5. HE scores were recorded for evaluation of the pathological changes. Blue arrows, normal tissue; red arrows, degenerative neurons; and white arrows, microglia. (B) Heat map shows the mean values of expression level of inflammatory cytokines in the brain at 0.5, 4, and 8 dpi after viral infection by the bio-plex assay, mean (n = 5). *p < 0.05, **p < 0.01, and ***p < 0.001, vs. normal control group (mock); ###p < 0.001, vs. WT-8 dpi group.

Figure 6. Pathological changes and pro-inflammatory factor expression in lung tissues caused by virus infection. (A) HE staining of lung tissue after virus infection at 3, 4, and 8 dpi, 400×, n = 3–5. HE scores were recorded for evaluation of the pathological changes. Grey arrows, normal tissue; red arrows, alveolar septal infiltration of inflammatory cells, infiltration of inflammatory cells around the bronchus; yellow arrows, alveolar epithelial hyperplasia and alveolar septum widening; green arrows, suspected virus inclusion body; black arrows, alveolar hyaline membrane; orange arrows, trachea epithelial ulcer; and blue arrows, alveolar interstitial exudation. **p < 0.01, vs. WT-4 dpi group. (B) Heat map shows the expression of inflammatory cytokines in the lung at 0.5, 4, and 8 dpi after infection with virus by the bio-plex assay, mean (n = 3–5); *p < 0.05, **p < 0.01, and ***p < 0.001, vs. normal control group (mock); #p < 0.05, ##p < 0.01, and ###p < 0.001, vs. WT-8 dpi group.

Figure 7. QJM increased the survival rate of P9-infected suckling mice and inhibited the nucleocapsid gene expression. After inoculation with an adapted-strain virus (P9) and treated with ARB and QJM, body weight change (A) and survival rate (B) of suckling mice within 15 days (n = 10) and the copy number of viral n gene in the brain and lungs at 4 dpi (C; n = 3–5). The data for viral n gene expression (copies/g) are presented as log10, mean ± SEM, and dashed lines indicate the detection limit.

Figure 8. QJM inhibited the N protein expression. N protein expression in brain (400×; A) and lung (400×; B) tissues (n = 3–5) was examined immunohistochemically with DAB staining at 4 dpi. The OC43 N protein expression level was calculated and indicated as H-Scores. Positive expression is shown in tan. Blue arrows, bronchus. *p < 0.05, **p < 0.01, and ***p < 0.001, vs. P9 group; #p < 0.05 and ###p < 0.001, vs. ARB group.

Figure 9. QJM therapy improved the pathological changes in the brain and lungs of P9-infected suckling mice. HE staining shows the pathological changes in brain (400×; A) and lung (200×; B) tissues at 4 dpi (n = 3–5). HE scores were recorded for evaluation of the pathological changes. Blue arrows, normal tissue; red arrows, progressive degeneration of neurons; white arrows, microglia; and black arrows, diseased tissue. *p < 0.05 and **p < 0.01, vs. P9 group.

Figure 10. QJM therapy modulated the expression of inflammatory/chemokines in the brain and lungs of P9-infected suckling mice. Heat map: The expression of inflammatory cytokines in the brain (A) and lung (B) at 4 dpi after treating with ARB and QJM by the bio-plex method, mean (n = 3–5); *p < 0.05, **p < 0.01, and ***p < 0.001, vs. P9 group; and #p < 0.05, ##p < 0.01, and ###p < 0.001, vs. ARB group.