Impact of different NA on initiation of influenza virus infection.

<p>MDCK cells were infected at an MOI of 0.001 in the presence of 1 µg/ml TPCK-trypsin. After adsorption for 1 h at 37°C, the inocula were removed and the cultures were washed 3 times. The cells were incubated at 37°C for 6 h. At the indicated time, the cells were processed for immunofluorescence, and the infected cells were detected with polyclonal antisera to whole viruses. (A) Fluorescence images of the infected cells at 6 h p.i. Fluorescent photomicrographs showing the intracellular expression of virus protein in cell culture. The FITC-fluorescence signal was expressed as the infected cells. (B) Volocity Demo software analysis of the ratios of infected cells according to the Fig. 5A. *, statistical significance (<i>p</i><0.05) (C) Flow-cytometric analysis of virus-infected cells at 6 h p.i. MDCK cells (2×10⁶) in suspension were incubated with PBS or anti-PR8 antibodies on ice. Then the FITC-conjugated IgG secondary antibodies were added. After washing, the cells were fixed and the number of infected cells was determined by flow cytometric analysis. *, statistical significance (<i>p</i><0.05).</p

Virus elution in <i>vitro</i>.

<p>50 µl two-fold dilutions of virus containing the HA titers of 1∶128 was incubated with 50 µl 0.5% chicken erythrocytes in microtiter plates at 4°C for 1 h. Then microtiter plates were incubated at 37°C, and the reduction of HA titers was measured periodically for 8 h.</p

Alignment of the deduced amino acid sequences of the NA genes from the influenza virus strains A/Chicken/Jiangsu/7/2002(H9N2), A/California/04/2009 (H1N1), A/chicken/Henan/12/2004 (H5N1) and PR8 (H1N1).

<p>Alignment of the deduced amino acid sequences of the NA genes from the influenza virus strains A/Chicken/Jiangsu/7/2002(H9N2), A/California/04/2009 (H1N1), A/chicken/Henan/12/2004 (H5N1) and PR8 (H1N1).</p

Influenza virus induced cell-cell fusion. MDCK cells were infected with the viruses at MOI of 0.1 or 0.001 in the presence of 1 µg/ml TPCK-trypsin.

<p>After adsorption for 1 h at 37°C, the inocula were removed and the cultures were washed 3 times. The cells were incubated for the indicated times at 37°C in the maintenance media. At the indicated time, the cells were processed for indirect immunofluorescence assay, and the infected cells were detected with polyclonal antisera to whole viruses. (A) MOI at 0.1, 3 h p.i. (B) MOI at 0.1, 6 h p.i. (C) MOI at 0.001, 12 h p.i.</p

Pathogenicity of recombinant viruses in BALB/c mice.

<p>Survival rates (A) and bodyweight changes (B) after challenge with the viruses. BALB/c mice were intranasally inoculated with rPR8-H5N1NA, rPR8-H9N2NA, rPR8-H1N1NA or PR8-wt virus at 1×10^6.5 EID₅₀. The survival rates and bodyweights of five mice in each group were measured daily from the date of challenge to 14 days after challenge. Values represent mean ± SD of each group of mice.</p

Replication of recombinant viruses in mice^a.

a<p>BALB/c mice were intranasally inoculated with recombinant or wild-type virus at 1×10^6.5 EID₅₀. On days 3 and 6 after infection, five mice from each group were killed for virus titration. Results are expressed as means ± SD.</p>b<p>ND, Not done.</p

Virus strains generated by reverse genetics and the amino acids comparison of NA^a.

a<p>The amino acids homology of wild-type PR8 virus and those of the H5N1, H9N2 or swine-H1N1.</p

Bone marrow MCPIP1 deficiency caused hematological disturbance.

<p><b>A</b>. Giemsa stained blood smears showed MCPIP1−/− bone marrow cell recipients contain dramatically reduced number of red blood cells. <b>B</b>. Vetscan HMT blood cell analysis showed reduced blood red cell (RBC) number, hemoglobin (HGB) concentration and hematocrit (HCT), as well as increased red cell distribution width (RDWc) which is calculated as Standard deviation ÷ mean cell volume x 100. <b>C</b>. Blood leukocyte profile showed increased number of neutrophils in MCPIP1−/− bone marrow cell recipients. <b>D.</b> Platelet related parameters of mice obtained by Vetscan HMT analysis. Parameters include total blood platelet count, platelet hematocrit (PCT), mean platelet volume (MVP), and platelet distribution width (PDWc) which is calculated using equation RDWc(%)  =  (Standard deviation ÷ mean cell volume) x 100. N = 6 (WT) or 9 (MCPIP1−/−).</p

Bone marrow MCPIP1 deficiency resulted in disorganization of mouse immune organs and leukocyte profile alteration in the spleen.

<p>Light microscopic images of mouse thymus, lymph nodes and spleen which were stained with hematoxylin and eosin. Magnification: 4X.</p

MCPIP1 deficiency increased macrophage cholesterol efflux.

<p><b>A</b>. Bone marrow-derived macrophages were loaded with ac-LDL and cholesterol efflux to DMEM, apoAI and HDL was measured. Data were presented as mean ± SEM. N = 3 in each group. <b>B</b>. Bone marrow-derived macrophages were cultured in DMEM for 48 h with or without ac-LDL. Cell lysates were used for western blotting analysis for ABCA1 and ABCG1. Representative western blotting result was shown. <b>C</b>. Quantitative analysis of the western blots. Data were presented as mean ± SEM. N = 4 in each group. Open column: WT macrophages; Black column, MCPIP1−/− macrophages.</p