Differential host determinants contribute to the pathogenesis of 2009 pandemic H1N1 and human H5N1 influenza A viruses in experimental mouse models

Abstract

Influenza viruses are responsible for high morbidities in humans and may, eventually, cause pandemics. Herein, we compared the pathogenesis and host innate immune responses of a seasonal H1N1, two 2009 pandemic H1N1, and a human H5N1 influenza virus in experimental BALB/c and C57BL/6J mouse models. We found that both 2009 pandemic H1N1 isolates studied (A/Hamburg/05/09 and A/Hamburg/NY1580/09) were low pathogenic in BALB/c mice [log mouse lethal dose 50 (MLD(50)) >6 plaque-forming units (PFU)] but displayed remarkable differences in virulence in C57BL/6J mice. A/Hamburg/NY1580/09 was more virulent (logMLD(50) = 3.5 PFU) than A/Hamburg/05/09 (logMLD(50) = 5.2 PFU) in C57BL/6J mice. In contrast, the H5N1 influenza virus was more virulent in BALB/c mice (logMLD(50) = 0.3 PFU) than in C57BL/6J mice (logMLD(50) = 1.8 PFU). Seasonal H1N1 influenza revealed marginal pathogenicity in BALB/c or C57BL/6J mice (logMLD(50) >6 PFU). Enhanced susceptibility of C57BL/6J mice to pandemic H1N1 correlated with a depressed cytokine response. In contrast, enhanced H5N1 virulence in BALB/c mice correlated with an elevated proinflammatory cytokine response. These findings highlight that host determinants responsible for the pathogenesis of 2009 pandemic H1N1 influenza viruses are different from those contributing to H5N1 pathogenesis. Our results show, for the first time to our knowledge, that the C57BL/6J mouse strain is more appropriate for the evaluation and identification of intrinsic pathogenicity markers of 2009 pandemic H1N1 influenza viruses that are "masked" in BALB/c mice.

Figure 1

Pathogenicity in BALB/c and C57BL/6J mice. BALB/c (A and B) or C57BL/6J (C and D) mice were infected with 10⁵ PFU of seasonal H1N1 (n = 14), HH05 (pH1N1) (n = 21), and HH15 (pH1N1) (n = 21) and with 10² PFU of human H5N1 (n = 20) and observed for survival and weight loss for 14 days. Survival and weight loss are presented for infected BALB/c (A and B, respectively) and C57BL/6J (C and D, respectively) mice. Control groups received PBS.

Figure 2

Organ tropism in BALB/c and C57BL/6J mice. BALB/c (A) or C57BL/6J (B) mice were infected with 10⁵ PFU of seasonal H1N1, HH05, and HH15 (pH1N1) and with 10² PFU of human H5N1. Virus titers in lung, brain, and gut homogenates of three animals per time point were determined by plaque assays on day 3 or 6 p.i. No virus was detected in uninfected control groups. The statistical significance of differences in virus titers was calculated by the Student's t-test (*P < 0.05 and **P < 0.01).

Figure 3

Lung pathological features in BALB/c and C57BL/6J mice. Mice were infected with 10⁵ PFU of seasonal H1N1 (B and G), HH05 (C and H), and HH15 (D and I) (pH1N1) and with 10² PFU of human H5N1 (E and J). Control groups received PBS (A and F). IHC stainings were performed from the lungs of three infected BALB/c (top) and C57BL/6J (bottom) mice obtained on day 6 p.i. using a polyclonal antibody, as previously described. Counterstaining with hematoxylin was performed as previously described. Antigen-positive cells are red-brown. Arrows indicate some infected cells; and asterisks, largely destructed and infiltrated areas. Original magnification, ×400 (all images were obtained using a light microscope).

Figure 4

Cytokine response in BALB/c and C57BL/6J mice. BALB/c and C57BL/6J mice were infected with 10⁵ PFU of seasonal H1N1, HH05, and HH15 (pH1N1) and with 10² PFU of human H5N1. Control groups received PBS. Levels were measured for Th1 cytokines (TNF-α, IFN-γ, and MCP-1) (A and C) and Th2 cytokines (IL-4, IL-6, and IL-10) (B and D) in BALB/c (gray bars) and C57BL/6J (black bars) mice by enzyme-linked immunosorbent assay. Bars represent mean ± SD cytokine concentrations of lung homogenates (A and B) from three animals or pooled serum samples (C and D) from four to seven animals on day 6 p.i. n.d. indicates not detected (ie, cytokine levels less than the detection limit). The statistical significance of differences in cytokine levels between BALB/c and C57BL/6J mice was calculated by the Student's t-test (*P < 0.05 and **P < 0.01).