Glycosylation on hemagglutinin affects the virulence and pathogenicity of pandemic H1N1/2009 influenza A virus in mice

Abstract

The two glycosylation sites (Asn142 and Asn177) were observed in the HA of most human seasonal influenza A/H1N1 viruses, while none in pandemic H1N1/2009 influenza A (pH1N1) viruses. We investigated the effect of the two glycosylation sites on viral virulence and pathogenicity in mice using recombinant pH1N1. The H1N1/144 and H1N1/177 mutants which gained potential glycosylation sites Asn142 and Asn177 on HA respectively were generated from A/Mexico/4486/2009(H1N1) by site-directed mutagenesis and reverse genetics, the same as the H1N1/144+177 gained both glycosylation sites Asn142 and Asn177. The biological characteristics and antigenicity of the mutants were compared with wild-type pH1N1. The virulence and pathogenicity of recombinants were also detected in mice. Our results showed that HA antigenicity and viral affinity for receptor may change with introduction of the glycosylation sites. Compared with wild-type pH1N1, the mutant H1N1/177 displayed an equivalent virus titer in chicken embryos and mice, and increased virulence and pathogenicity in mice. The H1N1/144 displayed the highest virus titer in mice lung. However, the H1N1/144+177 displayed the most serious alveolar inflammation and pathogenicity in infected mice. The introduction of the glycosylation sites Asn144 and Asn177 resulted in the enhancement on virulence and pathogenicity of pH1N1 in mice, and was also associated with the change of HA antigenicity and the viral affinity for receptor.

Figure 1. HA sequence alignment and examination of glycosylation.

(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.

Figure 2. Virus elution from erythrocytes.

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.

Figure 3. Characterization of viral growth in embryonated SPF chicken eggs.

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.

Figure 4. Weight loss and lung virus titres in infected mice.

(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.

Figure 5. The levels of antiviral and proinflammatory cytokines in lung of infected mice.

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.

Figure 6. Histopathologic changes of lung from H1N1 virus-infected mice.

Photomicrographs of hematoxylin-and-eosin strained lung sections from mice at 7 days p.i. are shown. Mice were infected with mock (A), H1N1/WT (B), H1N1/144 (C), H1N1/177 (D), H1N1/144+177 (E).

Figure 7. Structural locations of mutations found in pseudorevertant viruses.

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).