Variations in the hemagglutinin of the 2009 H1N1 pandemic virus: potential for strains with altered virulence phenotype?

Abstract

A novel, swine-origin influenza H1N1 virus (H1N1pdm) caused the first pandemic of the 21st century. This pandemic, although efficient in transmission, is mild in virulence. This atypical mild pandemic season has raised concerns regarding the potential of this virus to acquire additional virulence markers either through further adaptation or possibly by immune pressure in the human host. Using the mouse model we generated, within a single round of infection with A/California/04/09/H1N1 (Ca/04), a virus lethal in mice--herein referred to as mouse-adapted Ca/04 (ma-Ca/04). Five amino acid substitutions were found in the genome of ma-Ca/04: 3 in HA (D131E, S186P and A198E), 1 in PA (E298K) and 1 in NP (D101G). Reverse genetics analyses of these mutations indicate that all five mutations from ma-Ca/04 contributed to the lethal phenotype; however, the D131E and S186P mutations--which are also found in the 1918 and seasonal H1N1 viruses-in HA alone were sufficient to confer virulence of Ca/04 in mice. HI assays against H1N1pdm demonstrate that the D131E and S186P mutations caused minor antigenic changes and, likely, affected receptor binding. The rapid selection of ma-Ca/04 in mice suggests that a virus containing this constellation of amino acids might have already been present in Ca/04, likely as minor quasispecies.

Figure 1. Generation of an H1N1pdm virus lethal for Balb/c mice.

A) % Body weight changes and B) % survival of Balb/c (n = 4) and DBA (n = 4) mice inoculated intranasally with 5.4×10⁵ TCID₅₀ of Ca/04. DBA-Balb/c corresponds to Balb/c mice infected with lung homogenates containing the first passage Ca/04 virus from DBA mice. C) Growth kinetics of Ca/04 and ma-Ca/04 in MDCK cells. Viruses were inoculated at a multiplicity of infection of 0.001. Supernatants were collected at the indicated time points and titrated in MDCK cells by TCID₅₀. D) Balb/c mice inoculated intranasally with 1.2×10⁴ TCID₅₀ of ma-Ca/04 or Ca/04. Lungs from infected mice (n = 3/time point) were collected at 3 and 6 dpi, homogenized and virus titers measured in MDCK cells by TCID₅₀. Results are expressed as log₁₀ TCID₅₀/gr of tissue. E, F, G) Balb/c mouse lungs were collected at 3 dpi as described in D and fixed in 10% formalin, embedded in paraffin and sectioned. Serial sections were stained with H&E and the images were captured at ×20 magnification. E) Mock infected lung, F), Ca/04-infected lung, G) ma-Ca/04-infected lung.

Figure 2. Replication and transmission of ma-Ca/04 in ferrets.

A) Four groups of ferrets consisting of 1 infected, 1 direct contact (DC), and 1 respiratory contact (RC) per group were infected with 10⁶ TCID₅₀ of ma-Ca/04 as described in materials and methods. The graph represents the average ± SD of virus shedding (log₁₀ TCID₅₀/ml of nasal wash) over time (in days, dpi) of inoculated (infected, grey open squares), direct contacts (orange triangles), and respiratory contact ferrets (purple squares). B) Two groups of ferrets consisting of 1 infected, 1 direct contact (DC), and 1 respiratory contact (RC) per group were infected with 10⁶ TCID₅₀ of Ca/04 as described in materials and methods. The graph represents the average ± SD of virus shedding (log₁₀ TCID₅₀/ml of nasal wash) over time (in days, dpi) of inoculated (infected, blue open squares), direct contacts (green diamonds), and respiratory contact ferrets (red dots). Transmission was monitored by titrating the amount of virus in the nasal washes of ferrets collected daily. BLD, below limit of detection (0.699 log₁₀ TCID₅₀/ml). C) Two ferrets/group were infected with 10⁶ TCID₅₀ of either Ca/04 or ma-Ca/04. Infected ferrets were euthanized at 4 dpi and brains, olfactory bulbs nasal turbinates, tracheas, and lungs were collected, homogenized and titrated in MDCK cells by TCID₅₀. Results show log₁₀ TCID₅₀/gr of tissue.

Figure 3. Structural analysis of amino acid mutations on the HA of ma-Ca/04.

A) Side view of the H1 HA trimeric structure based on A/South Carolina/1/1918 (H1N1) (PDB code: 1RUZ) showing the 5 major antigenic sites on the globular head of the molecule with respect to amino acid changes between the ma-Ca/04 and Ca/04 viruses. RBS, receptor-binding site (dark blue); 131, D131E mutation (red); 186, S186P mutation (green); Sa, antigenic site A (cyan); Sb, antigenic site B (light orange); Ca1, antigenic site C2a (purple); Ca2, antigenic site C2a (olive); Cb, antigenic site Cb (lemon). B) Top view of H1 HA trimer as described in A); 198, A198E mutation (yellow) present on Sb. C) Close-up of the RBS in one of the HA monomers, highlighting amino acids involved in receptor binding (138, 190, 194, 225, 226, and 228, in blue) with respect to amino acid mutations between ma-Ca/04 and Ca/04 (D131E, red; S186P, green - part of the RBS -; and A198E, yellow). Structures generated using MacPymol (DeLano Scientific).

Figure 4. Effects of amino acid mutations for virulence of ma-Ca/04.

A) Polymerase activity measured with mini-genome assay. Each transfection consisted of 6 plasmids encoding a minigenome influenza replicon (Gluc flanked by influenza NS gene untranslated regions), PB1, PB2, PA, NP encoding plasmids from Ca/04 (or ma-Ca/04 as indicated) and SEAP (used to normalize transfection efficiency). Relative activity calculated as the fold difference in the ratio of Luc/SEAP activity as described in materials and methods. Data corresponds to three independent experiments with samples run in duplicates. The maPA corresponds to transfection of the ma-Ca/04 PA gene, the maPA-NP corresponds to transfection of the ma-Ca/04 PA and ma-Ca/04 NP genes, the maNP corresponds to transfection of the ma-Ca/04 NP gene, the caPA-NP corresponds to transfection of the Ca/04 PA and Ca/04 NP genes, and pDP2002 corresponds to empty vector control, respectively, in the minigenome assay. B) Growth kinetics of recombinants of Ca/04 carrying either HA (maHA1:7Ca/04), HA and PA (maHA-PA2:6Ca/04), HA and NP (maHA-NP2:6Ca/04), or HA, PA, and NP (maHA-PA-NP3:5Ca/04) from ma-Ca/04. Viruses were inoculated at a multiplicity of infection of 0.001. Supernatants were collected at the indicated time points and titrated in MDCK cells by TCID₅₀. C) % body weight over time in Balb/c mice (n = 3) infected with viruses produced in B) (1.2×10⁵ TCID₅₀/mouse). D) % body weight over time in Balb/c mice (n = 3) infected with recombinant viruses of NL/602 carrying either the wt HA from NL/602 (NL/602), ma-Ca/04 HA (maHA1:7NL/602) or NY/18 HA (8.0×10⁴ TCID₅₀/mouse). E) and F) % body weight over time in Balb/c mice (n = 5) infected with recombinants carrying single, double or triple mutations in HA in the backbone of Ca/04 (8.0×10⁴ TCID₅₀/mouse). G) Mice (n = 6/virus) inoculated intranasally with mutants produced in E) (8.0×10⁴ TCID₅₀/mouse). Lungs from infected mice (n = 3/time point) were collected at 3 and 6 dpi, homogenized and virus titers measured in MDCK cells by TCID₅₀. Results are expressed as log₁₀ TCID₅₀/gr of tissue.