Mouse-adapted H9N2 influenza A virus PB2 protein M147L and E627K mutations are critical for high virulence

Abstract

H9N2 influenza viruses have been circulating worldwide in multiple avian species and have repeatedly infected humans to cause typical disease. The continued avian-to-human interspecies transmission of H9N2 viruses raises concerns about the possibility of viral adaption with increased virulence for humans. To investigate the genetic basis of H9N2 influenza virus host range and pathogenicity in mammals, we generated a mouse-adapted H9N2 virus (SD16-MA) that possessed significantly higher virulence than wide-type virus (SD16). Increased virulence was detectable after 8 sequential lung passages in mice. Five amino acid substitutions were found in the genome of SD16-MA compared with SD16 virus: PB2 (M147L, V250G and E627K), HA (L226Q) and M1 (R210K). Assessments of replication in mice showed that all of the SD16-MA PB2, HA and M1 genome segments increased virus replication; however, only the mouse-adapted PB2 significantly increased virulence. Although the PB2 E627K amino acid substitution enhanced viral polymerase activity and replication, none of the single mutations of mouse adapted PB2 could confer increased virulence on the SD16 backbone. The combination of M147L and E627K significantly enhanced viral replication ability and virulence in mice. Thus, our results show that the combination of PB2 amino acids at position 147 and 627 is critical for the increased pathogenicity of H9N2 influenza virus in mammalian host.

Figure 1. Viral growth kinetics of SD16 and SD16-MA in MDCK and A549 cells.

Confluent MDCK (A) or A549 (B) cells were infected with H9N2 viruses at an MOI of 0.01. Virus yields at 12, 24, 36, 48, and 60 hpi were titrated in MDCK cells. Each data point represents the mean virus yield from three individually infected wells ± SD.

Figure 2. Virulence and death pattern of mice infected with different H9N2 viruses.

Five-week-old BALB/c mice (five/group) were inoculated intranasally with different H9N2 viruses, SD16 (A), SD16-MA (B), SD16:MA PB2 (C), or SD16-M147L/E627K (D). Doses of 10⁴ to 10⁶ pfu (A) or 10² to 10⁵ pfu (B, C, D) were used.

Figure 3. Virulence of SD16 and SD16-MA viruses in mice.

Groups of five mice were inoculated intranasally with 10⁵ pfu of SD16 or SD16-MA virus. Body weight (A) and survival (B) were monitored daily for 14 dpi. (C) Histopathology of SD16 and SD16-MA infected mice. Lungs were collected at 5 dpi and fixed in 10% formalin, embedded in paraffin and sectioned. SD16 caused mild and limited alveolitis, whereas SD16 induced severe interstitial pneumonia, including diffuse injury of lungs, alveolar collapse, red blood cells and immune cells infiltration. (D) Virus titers in lungs of infected mice. Groups of three mice were inoculated intranasally with 10⁵ pfu of SD16 or SD16-MA viruses and lungs were collected at 3 dpi for virus titration in MDCK cells. Each value represents mean viral yield from three individually infected lungs ± SD. *, P<0.05 compared with the value of SD16.

Figure 4. Cytokine responses in lungs of infected mice.

Groups of three BALB/c mice were inoculated intranasally with 10⁵ pfu of SD16 or SD16-MA virus. The lungs of mice were collected at 3 dpi. The concentrations of various cytokines in lung homogenates were measured by a protein array analysis with Bio-Plex Mouse Cytokine 8-Plex. Each value represents mean cytokine concentration of three mice from each infected group ± SD. *, P<0.05 compared with the value of SD16 infected mice.

Figure 5. Amino acid differences between SD16 and SD16-MA.

The amino acid location of mutations are numbered and indicated with arrowheads on the linear sequence. The locations of regions of protein binding, or functions are indicated with rectangles and are labeled with respect to interacting viral proteins. The PB1, NP and RNP ribonucleocapsid protein binding regions are in purple, green and yellow respectively; PB2 cap binding regions are in orange; HA receptor sites are in red.

Figure 6. Viral RNA polymerase activity and Viral growth kinetics in MDCK and A549 cells.

(A) Polymerase activity of SD16 with different PB2 mutations in a minigenome assay. Four protein expression plasmids (PB2, PB1, PA, NP) for the RNP combinations were transfected into 293T cells together with luciferase reporter plasmid pYH-Luci and internal control plasmid Renilla, as described in Materials and Methods. The values shown are means ± SD of results for three independent experiments and are standardized to the activity of SD16 (100%). *, P<0.05 compared with that of SD16-infected cells. (B, C, D, E) Viral growth kinetics of reassortant viruses and PB2 mutants in MDCK and A549 cells. Confluent momolayers of cells were infected with rescued H9N2 viruses at an MOI of 0.01. Cell supernatants were harvested every 12 hours until 60 hpi and titrated by plaque assay on MDCK cells. Each data point represents the mean virus yield from three individually infected wells ± SD. (B) reassortant viruses in MDCK cells, (C) PB2 mutants in MDCK cells, (D) reassortant viruses in A549 cells, (E) PB2 mutants in A549 cells.