Figure 1.
Body weight changes (a) and survival rate (b) of the BALB/c mice infected with 106 PFU of A/chicken/Zhejiang/DTID-ZJU01/2013(H7N9) (CK1, *) or 105 PFU of A/Anhui/1/2013(H7N9) (AH1, ⋄) via intranasal route.
Body weight and survival were monitored for 14 days after virus infection. Data shown are the average of three experiments (n = 17 for CK1 and n = 20 for AH1 group).
Figure 2.
Histopathological changes in the lung tissue infected with CK1 or AH1.
Representative histological images of haematoxylin and eosin (H&E) stained lung tissue sections of normal mouse lung (a, and amplified image b) and infected mouse lung at various time points post infection (c-h). Mouse lung at day 2 p.i. showed peribronchiolar interstitial infiltration, bronchiole epithelial cell necrosis and necrotic cell debris within alveolar lumens (c, CK1 infection; d, AH1 infection). At day 4 p.i, mouse lung showed alveolar space exudation, bronchiole epithelial cell necrosis, alveolar cell necrosis and destruction of alveolar wall (e and f). At day 6 p.i., alveolar exudation, infiltration, hyaline membrane formation and alveolar hemorrhage with red blood cells within the alveolar space (g and h). Original magnification ×100.
Table 1.
Average histological score of CK1- and AH1-infected mouse lung tissues*.
Figure 3.
CK1 and AH1 replication profile in mouse lung.
A. Representative images of immunohistochemically stained influenza nucleoprotein (NP) in formalin fixed mouse lung tissue infected with CK1 or AH1 at day 2 p.i. Viral NP protein was labeled brown by 3,3′-diaminobenzidine (DAB). Uninfected mouse lung as negative control (a), amplified image (b); Representative images of CK1(c) and AH1 (d) infected mouse lung stained NP positive at 40x magnification. Trachea epithelial cells (e and f), bronchiole epithelial and alveolar epithelial cells (g and h) were stained positive for viral NP protein. (Original magnification ×200). B. Viral load in infected mouse lung homogenates. Mice were infected with 105 of AH1 or 106 PFU of CK1, at day 1, 2, 4 and 6 p.i., 3–5 mice from each group were sacrificed. The left side of the lung was homogenized in 1 ml of MEM culture medium. Viral loads were determined by amplification of viral M gene copy numbers by real time RT-PCR (top panel), and infectious viral titre were determined by TCID50 assay on MDCK cells (bottom panel). **P<0.01; * P<0.05.
Figure 4.
Liver, kidney and heart tissue degenerative changes in CK1-infected mice at day 6 post infection.
Representative images of haematoxylin and eosin (H&E) stained tissue sections are presented. The liver (a), kidney (d), heart (g) of uninfected mice were shown for comparison. CK1-infected mice at day 6 p.i. showed liver hepatocytes degeneration, focal cells necrosis (arrows, b) and hemorrhagic changes (c); kidney tubular epithelial cells degenerative changes (arrows, e) and peritubular vessels congestion (f). Mild myocardial cell swelling (arrows, h) and red blood cells infiltrating between degenerative myocardial fibers (i) were seen in the heart. Original magnification ×200.
Figure 5.
Cytokines and COX-2 expression in infected mice lung and mouse lung epithelial cell line LA-4.
(A) The protein levels of cytokines IL-1β, IL-6, RANTES and IL-10 presented in mouse lung homogenates were determined by ELISA. On day 1, 2, 4 and 6 p.i., the left lungs from infected mice (3–5 mice from each group) were homogenized in 1 ml of MEM medium. Clarified homogenates were used for cytokine detection. Non-infected mouse lung specimens were used as baseline controls. (B) mRNA level of IFN-α, IFN-β, IFN-γ and COX-2 genes in mouse lung were determined by real time RT-PCR. Mouse β-actin mRNA was used for RNA concentration normalization. Error bar indicates ±SD. * P<0.05. C. viral load, IL-6 and COX-2 mRNA levels in CK1- and AH1-infected mouse lung epithelial cell line LA-4 determined by real time RT-PCR. 2×105 cells/per well in 12-well plate were infected with AH1 or CK1 at M.O.I of 2. At indicated times post virus infection, the cells were harvested for RNA extraction and real time RT-PCR detection of viral M gene (top panel) and IL-6 (middle panel) and COX-2 (bottom panel) mRNA. β-actin was used as RNA concentration normalization, and the data presented are the average of two experiments. * P<0.05.
Figure 6.
Survivals and body weight changes of mice treated with combination of zanamivir and celecoxib, zanamivir or celecoxib alone by intraperitoneal injection.
The mice were infected with 106 PFU of CK1 and treated with (1) combination treatment (♦): celecoxib 2 mg daily from day 2 to day 4 p.i., and zanamivir 2mg twice daily from day 2 to day 8 p.i; (2) zanamivir alone (Δ): zanamivir 2mg twice daily from day 2 to day 8 p.i; (3) celecoxib alone (▾): celecoxib 2 mg daily from day 2 to day 4 p.i., (4) control group (×): Celecoxib solven (1% DMSO/PBS) 200ul from day 2 to day 4 p.i. The survival (a) and body weight change (b) were observed for 14 days after infection. Data shown are the average of two experiments (in total, n = 10 for each treatment group, n = 17 for control group). *P<0.05 as compared to untreated control group.
Figure 7.
Changes of cytokines and viral loads in the lungs of CK1-infected mice after treatments.
At day 4 p.i., mice from different treatment groups were sacrificed. Left-side lungs were homogenized and the clarified homogenate were used for IL-6, IL-1β and RANTES determination by EIA (a) and the viral loads were determined by real time quantitative RT-PCR (b). n = 5 for each group. Error bar indicates ±SD. (c) Representative histological images of H&E stained lung tissues of untreated mice (top left panel), treated with celecoxib-zanamivir combination (top right panel), zanamivir alone (bottom left panel), and celecoxib alone (bottom right panel). Original magnification ×100. (d) Average histology score of mouse lung tissues with different treatment.
Table 2.
Mouse lung tissue histological score.