Figure 1.
Growth kinetics of hMPV and RSV in A549 cells and mouse lungs.
A) A549 cells were infected with RSV or hMPV at an MOI of 0.1. At the indicated time points the virus-containing media were collected and determined for the viral titer by plaque assay. Viral titer was expressed as log PFU/ml culture media. * P<0.05 vs. hMPV titer on the corresponding time points. B) Eight mice in each group were infected with 1.5×106 PFU hMPV or RSV. At the indicated time points post infection, the mice were sacrificed. Mouse lungs were homogenated and then subjected to viral titration by the plaque assay on Vero E6 cells and Hep-2 cells. Viral titer was expressed as log PFU/g lung tissue. * P<0.05 vs. hMPV titer on the corresponding time points.
Figure 2.
Lung histopathology of infected mice on day 5 after hMPV or RSV infection.
Eight mice in each group were sacrificed on day 5 post-infection and lungs were harvested, fixed with 4% paraform, embedded in paraffin, cut into sections and stained with haematoxylin-eosin (H and E). A) Representative lung section, magnification×200. B) The histopathologic score (HPS) was determined and data were expressed as mean ±SD (n=6). * P<0.05 vs. control, § P<0.05 vs. hMPV-infected mice.
Figure 3.
The mRNA expressions of TLRs in A549 cells after hMPV or RSV infection.
A549 cells were inoculated by hMPV, RSV, UV-MPV or UV–RSV at an MOI of 2, and cells were harvested at 3, 6, 9, 12 and 24 h after infection for total RNA extraction. A) The TLR1-10 mRNA levels were detected by RT-PCR at 6 h after hMPV or RSV infection. Values of TLR1-10 mRNA/beta-actin mRNA were expressed as mean ±SD (n=6). Normal A549 cell served as the MOCK. * P<0.05 vs. MOCK. B) to F) were the dynamic expression of TLR3-4, TLR7-9 respectively. The TLR3-4 and TLR7-9 mRNA levels were detected by real time RT-PCR at 3, 6, 9, 12 and 24 h after inoculated by hMPV, RSV, UV-MPV or UV–RSV. β-actin served as an internal reference, and untreated cells as control. Data were expressed as mean ±SD (n=6). * P<0.05 vs control.
Figure 4.
Messenger RNA expressions of TLRs in the lungs of BALB/c mice after hMPV or RSV infection.
Mice in HMPV group, RSV group and control group were sacrificed at 1, 3, 5, 7, 9 and 16 days after infection, and the lungs were harvested for extraction of total RNA and determination of mRNA expressions of TLR3-4 and TLR7-8 by real-time PCR. β-actin served as an internal reference. Data were expressed as mean ±SD (n=8). * P<0.05 vs. control; § P<0.05 vs. hMPV group. A) to D) were the expression of TLR3-4, TLR7-8 respectively.
Figure 5.
Expression of TLR3 protein intracellular or on the surface of A549 cells by flow cytometry.
A549 cells were pre-treated with Poly(I:C) (10 µg/mL) for 6 h, then infected by hMPV or RSV at an MOI of 2 at 37°C for 24h. Cells were harvested for assessing TLR3 expression by flow cytometry. Cell surface and intracellular staining were shown. TLR3 protein was located mainly intracellularly. A) Red histograms represent staining with the specific antibody underlaid with the isotype-matched control antibody (green histograms). B) Data were expressed as mean ±SD (n=6). * P<0.05 vs. mock; § P<0.05 vs. hMPV.
Figure 6.
Secretion of IFN-α, TNF-α and IL-8 from A549 cells after infection of RSV or hMPV or pretreated with PolyI:C.
A549 cells were seeded in a 6-well plate, and culture medium was replaced with serum free DMEM on the second day. Some were pretreated with Poly(I:C) (10µg/mL) for 6 h, and then infected by hMPV and RSV at an MOI of 2 at 37°C for 24 h. The supernatants were collected and ELISA was performed to detect the production of IFN-α, TNF-α and IL-8. Data were expressed as mean ±SD (n=6). * P<0.05 vs. normal control; § P<0.05 vs. hMPV; a P<0.05 vs. PolyI:C+hMPV.