Differential host gene responses in mice infected with two highly pathogenic avian influenza viruses of subtype H5N1 isolated from wild birds in Thailand

Abstract

In Thailand, highly pathogenic avian influenza (HPAI) viruses of subtype H5N1 had been isolated from various wild birds during the HPAI outbreak in poultries. In this study, we examined the pathogenicity of two wild bird isolates (A/Pigeon/Thailand/VSMU-7-NPT/2004; Pigeon04 and A/Tree sparrow/Ratchaburi/VSMU-16-RBR/2005; T.sparrow05) in mice. They showed similar replication in several organs and lethal outcome. However, on day 3 post-infection, Pigeon04 induced mRNA expression of proinflammatory cytokines (IL6 and TNFα) and MIP-2, neutrophil chemoattractant, in the lungs, resulting in severe pneumonia that was accompanied by neutrophil infiltration. In contrast, on day 7 post-infection, T.sparrow05 induced the expression of several cytokines to a greater extent than Pigeon04; it also potently induced mRNA expression of several cytokines in brains of the infected mice that triggered frequent inflammatory events. In sum, our study demonstrated that two HPAI viruses induced different host responses, despite having similar replications, resulting in lethal outcome in mice.

Fig. 1

Growth properties of two H5N1 HPAI viruses in MDCK cells. MDCK cells were infected with the HPAI viruses at a MOI of 0.01 TCID₅₀ (3.7 log₁₀ TCID₅₀/ml). Supernatants were collected on days 1, 2 and 3 post-infection, and virus titers were determined on MDCK cells by TCID₅₀ assay. The data were indicated as mean virus titers ± standard deviations of three independent experiments. Asterisks indicate statistically significant differences (*p < 0.05 by Student t test).

Fig. 2

Lethality and viral shedding of the two H5N1 HPAI viruses isolated from wild birds in chickens. (A) Survival rate of White Leghorn chickens after intranasal inoculation with Pigeon04 and T.sparrow05 at a dose of 10⁶ EID_50. (B) Virus titers of trachea and cloacal swabs of dead chickens. The bars indicate the average for each group.

Fig. 3

Lethality and replication of the two H5N1 HPAI viruses isolated from wild birds in mice. (A and B) Survival rate of BALB/c mice infected with Pigeon04 (A) or T.sparrow05 (B) at the dose of 10⁰ to 10⁵ EID₅₀ (10⁰ EID₅₀, ■; 10¹ EID₅₀, ●; 10² EID₅₀, ♦;10³ EID₅₀, □; 10⁴ EID₅₀, ○; 10⁵ EID₅₀, ◊). (C and D) Viral distribution in mice infected with HPAI viruses. Mice were inoculated with Pigeon04 and T.sparrow05 at a dose of 10^3.5 and 10⁴ EID₅₀, respectively. Three mice in each group were sacrificed on days 3 and 7 post-inoculation for virus titration. Virus titers are presented as the mean values ± standard deviation. The dashed line indicates the detection limit of the virus titers. An asterisk indicates a statistically significant difference (*p < 0.05 by Student t test).

Fig. 4

Representative hematoxylin- and eosin-stained histopathological (A–D) and immunohistochemical (E–H) images of mice tissues infected with Pigeon04 (A, C, E and G) and T.sparrow05 (B, D, F and H). Lung sections of mice sacrificed on day 3 post-infection (A, B, E and F) and brain section of dead mice on day 8 post-infection (C, D, G and H) are shown. Immunohistochemistry detected viral antigen in the tissues. Arrowheads indicate neutrophil infiltrated into tissues. Original magnification: × 20 (A–D); × 40 (E–H).

Fig. 5

Comparison of host gene responses of the lungs in mice infected with Pigeon04 and T.sparrow05. Mice were inoculated with Pigeon04 or T.sparrow05 at the dose of 10^3.5 and 10⁴ EID₅₀, respectively. At the indicated time, three mice in each group were sacrificed, and RNA was extracted, as mentioned in Materials and methods. The level of mRNA expression of each gene was examined by real-time PCR analysis using primers specific to the corresponding gene. mRNA levels indicate mean values ± standard deviations. Statistical analysis was performed for the Pigeon04-infected, T.sparrow05-infected and uninfected groups by ANOVA followed by Turkey analysis. The sharps indicate that the virus-infected group was significantly (p < 0.05) different from the uninfected group. Asterisks indicate that the Pigeon04-infected group was significantly (p < 0.05) different from the T.sparrow05-infected group.

Fig. 6

Comparison of host gene responses of brains in mice infected with Pigeon04 and T.sparrow05. mRNA levels indicate mean values ± standard deviations. Sharps indicate that virus-infected group was significantly (p < 0.05) different from uninfected group. Asterisks indicated that Pigeon04-infected group was significantly (p < 0.05) different from T.sparrow05-infected group.