Substitution of D701N in the PB2 protein could enhance the viral replication and pathogenicity of Eurasian avian-like H1N1 swine influenza viruses

Figures & data

Fig. 1 Polymerase activity of the PB2 mutants in human cells.

293T cells were transfected with a pFluc plasmid expressing negative-sense virus-like RNA and encoding firefly luciferase (Fluc) and a pRluc plasmid expressing the Renilla luciferase gene (Rluc) as an internal control (Promega). The 293T cells were also co-transfected with plasmids expressing the wild-type (701D) and mutated (701 N) PB2, PB1, NP, and PA segments derived from the A/Hunan/42443/2015(H1N1) viruses. After being cultured at 37 °C for 24 h, cell lysates were used to measure the Fluc and Rluc activity levels. Mock transfection with the two reporter constructs only was set to 100%. The means ± SD of triplicate experiments are shown. **P < 0.01

Fig. 2 Infection of C57BL/6 mice with recombinant HuN EA-H1N1 viruses.

Eight- to ten-week-old female C57BL/6 mice (n = 5/group) were inoculated intranasally with 10¹ (a), 10² (b), 10³ (c), 10⁴ (d), 10⁵ (e), and 10⁶ TCID₅₀ (f) of the recombinant viruses. Mice receiving PBS were used as controls. Weight loss was monitored for 14 days

Fig. 3 Mortality of the recombinant HuN EA-H1N1 viruses in mice.

Eight- to ten-week-old female C57BL/6 mice (n = 5/group) were inoculated intranasally with 10¹ (a), 10² (b), 10³ (c), 10⁴ (d), 10⁵ (e), 10⁶ TCID₅₀ (f) of the recombinant viruses. Mice receiving PBS were used as controls. Survival was monitored for 14 days

Seroconversions of mice inoculated with recombinant viruses^a

Virus and dose, log10 TCID50/50 μl	Hemagglutination inhibition titer^b (HuN EA-H1N1 virus antigen)					MID50, log10 TCID50^c
	Mouse 1	Mouse 2	Mouse 3	Mouse 4	Mouse 5
rgHuN-PB2701D						1.5
6	ND	ND	ND	ND	ND
5	ND	ND	ND	ND	ND
4	640	640	640	640	640
3	320	320	640	640	640
2	20	≤10	640	320	≤10
1	<10	40	<10	160	320
rgHuN-PB2701N						0.5
6	ND	ND	ND	ND	ND
5	ND	ND	ND	ND	ND
4	ND	ND	ND	ND	ND
3	640	640	ND	ND	ND
2	640	640	640	ND	ND
1	640	160	40	320	320
PBS	<10	<10	<10	<10	<10

MID₅₀ 50% mouse infectious dose, ND not determined due to death of mice (treated as positive), TCID₅₀ 50% tissue culture infectious dose

^aMice serum was collected at 14 days post inoculation

^bAn HI titer of <40 was recognized as negative

^cMID₅₀ was determined by the Spearman–Karber method³²; TCID₅₀ was calculated using the Reed-Muench formula³³

Fig. 4 Replication of the recombinant viruses in the respiratory tracts of mice.

Eight- to ten-week-old female C57BL/6 mice (n = 3/time-point) were inoculated intranasally with 10³ TCID₅₀ (left) or 10⁵ TCID₅₀ (right) of the recombinant viruses. Animals were euthanized at 1 (a), 4 (b), and 7 (c) dpi. Viral titers in the tissues were determined on MDCK cells. NT Nasal turbinate, TR trachea, LU lung

Fig. 5 Replication and pathological changes in the lungs of mice inoculated with recombinant viruses.

Eight- to ten-week-old female C57BL/6 mice (n = 3/time-point) were inoculated intranasally with 10³ TCID₅₀ of the rgHuN-PB2_701D viruses (a, f) and rgHuN-PB2_701N viruses (c, h) or 10⁵ TCID₅₀ of the rgHuN-PB2_701D viruses (b, g) and rgHuN-PB2_701N viruses (d, i). Mice receiving PBS (e, j) were used as controls. Mice were killed at 4 dpi, and the lung lobes were used for NP antigen staining (upper panel) and hematoxylin and eosin (H&E) staining (lower panel)

Fig. 6 Replication kinetics of the recombinant viruses in wild-type (WT) and importin-α7 knockout (α7-KO) A549 cells.

Cells were inoculated with the recombinant viruses at 37 °C, and culture supernatants were harvested at 0, 24, 48, 72 and 96 h post-inoculation (hpi). Virus titers were determined on MDCK cells. a Growth curves of the rgHuN-PB2_701D viruses in WT (black, filled diamonds) and α7-KO (blue diamonds) A549 cells; b Growth curves of the rgHuN-PB2_701N viruses in WT (red, filled triangles) and α7-KO (green triangles) A549 cells; c Growth curves of the rgHuN-PB2_701D viruses (black, filled diamonds) and the rgHuN-PB2_701N viruses (red, filled triangles) in WT A549 cells; d Growth curves of the rgHuN-PB2_701D viruses (blue diamonds) and the rgHuN-PB2_701N viruses (green triangles) in α7-KO A549 cells. The results are presented as the means ± SD of three repeated experiments. *P < 0.05

Fig. 7 Sub-localization of vRNPs in wild-type (WT) and importin-α7 knockout (α7-KO) A549 cells infected with the recombinant viruses.

WT and α7-KO A549 cells were infected with recombinant viruses at 37 °C and were fixed at indicated time points (1.5 hpi early in the life cycle and 4 hpi in the middle of the life cycle). Cells were then immunostained with an antibody against NP and an Alexa Fluor 594-conjugated secondary antibody. Cell nuclei were stained with DAPI

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