Effect of variations at four HAs on reactivity with monoclonal antibodies.

<p><b>Note.</b>¹ The monoclonal antibodies(5D₅,4D₇,4E₁,2B₃,3G₁₂,1C₉,4B₁₂,2C₅,2H₇,5F₇) were specific against HA of pH1N1/WT. HI tests were performed by standard methods using chicken red blood cells. (NT), microneutralization assay.</p

Virus elution from erythrocytes.

<p>Two-fold dilutions of virus containing the HA titers of 1∶32 (2⁵) were incubated with equal volume of 0.5% chicken erythrocytes in microtiter plates at 4°C for 1 h. The microtiter plates were then stored at 37°C, and the reduction of HA titers was recorded periodically for 240 min.</p

Primers for real-time RCR.

<p><b>NOTE</b>. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IFN, interferon; IL, interleukin; MCP, monocyte chemotactic protein; TNF, tumor necrosis factor.</p

Structural locations of mutations found in pseudorevertant viruses.

<p>The antigenic site Sa is colored red. 144^th and 177^th sialic acids modeled as green stick structure. Images are derived from the crystal structure of the hemagglutinin of A/California/04/2009 H1N1 virus. Images were generated using the MacPyMol software and the solved HA structure (ID 3LZG; RCSB Protein Data Bank).</p

Characterization of viral growth in embryonated SPF chicken eggs.

<p>The eggs were infected with each of the viruses and were maintained at 37°C. The viral titers in the allantoic fluid of infected eggs were examined at 24, 48, 72, 96 h after infection by EID₅₀. Titers were expressed as log₁₀ EID₅₀.</p

HA sequence alignment and examination of glycosylation.

<p>(A) The HA protein sequence alignment of pandemic H1N1/2009 virus. (B) Examination of glycosylation of the H1N1 HA proteins by Western blotting. 1: H1N1/144, 2: H1N1/177, 3: H1N1/144+177 and 4: H1N1/WT.</p

The levels of antiviral and proinflammatory cytokines in lung of infected mice.

<p>Determination of IL-1, IL-10, MCP-1, TNF-α and IFN-γ levels in H1N1/144, H1N1/177, H1N1/144+177 or H1N1/WT-infected mouse lungs at 2, 5, 7, 9 d.p.i. The statistical analysis was performed using Student's t test. *, P<0.05; **, P<0.01 between the recombinant mutants and H1N1/WT.</p

Weight loss and lung virus titres in infected mice.

<p>(A) Weight loss of mice inoculated with strains having different N-glycosylation sites on HA. Six-week-old BALB/c mice were inoculated intranasal with 10³ 50% egg infectious dose (EID₅₀) of the recombinant influenza viruses, with 8 mice per group. P<0.05 between H1N1/144+177 and H1N1/WT on 3, 4 days post infection, P<0.01 between H1N1/144+177 and H1N1/WT on 5 days post infection, P<0.05 between H1N1/177 and H1N1/WT on 3–12 days post infection, P<0.01 between H1N1/177 and H1N1/WT on 13, 14 days post infection, P<0.01 between H1N1/144 or H1N1/144+177 and H1N1/WT on 6–14 days post infection. (B) Viral titres in lung of infected mice were indicated by the expression of NP gene. The statistical analysis was performed using Student's t test. *, P<0.05; **, P<0.01 between the recombinant mutants and H1N1/WT viral titer.</p

The glycosylation sites on HA in different types of A/H1N1 influenza viruses.

<p><b>NOTE.</b> The amino acid of potential glycosylation sites was T (144) and N (177) on HA.</p

Higher replication and IFNα induction in GM-DCs of the 6:2 Tky/05 virus depends on mammalian adaptation of RdRp and was not seen for virus with internal genes from PR8.

<p>Bone marrow derived GM-DCs were infected with the indicated virus at MOI = 10. Total RNA was harvested at 8 hpi. RT-PCR was performed using primers targeting segment 4 (HA) (A-C) and segment 7 (M) (D-G) respectively. Bars represent mean ± SD (n = 3). Differences between groups were analyzed with one-way ANOVA and Bonferroni’s multiple comparisons test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; unlabeled indicates no significant difference.</p