Assessment of Antiviral Properties of Peramivir against H7N9 Avian Influenza Virus in an Experimental Mouse Model

Abstract

The H7N9 influenza virus causes a severe form of disease in humans. Neuraminidase inhibitors, including oral oseltamivir and injectable peramivir, are the first choices of antiviral treatment for such cases; however, the clinical efficacy of these drugs is questionable. Animal experimental models are essential for understanding the viral replication kinetics under the selective pressure of antiviral agents. This study demonstrates the antiviral activity of peramivir in a mouse model of H7N9 avian influenza virus infection. The data show that repeated administration of peramivir at 30 mg/kg of body weight successfully eradicated the virus from the respiratory tract and extrapulmonary tissues during the acute response, prevented clinical signs of the disease, including neuropathy, and eventually protected mice against lethal H7N9 influenza virus infection. Early treatment with peramivir was found to be associated with better disease outcomes.

FIG 1

Pathogenicity of A/Shantou/1001/2014 H7N9 influenza virus in C57/BL6 mice. Animals (n = 10/group) were infected with different viral concentrations by the intranasal route, and weight loads (a) and mortality (b) were monitored until 14 dpi. Survival curves were found to be significantly different at each viral concentration (P < 0.0005).

FIG 2

Pathological changes in lung tissues infected with A/Shantou/1001/2014 H7N9 influenza virus. C57/BL6 mice were infected with 10⁴ EID₅₀ of H7N9 virus, and lung sections were stained with H&E. Lung sections at 3 dpi showed interstitial pneumonia with focalized lesions at the periphery (white arrow) (original magnification, ×40) (A), hemorrhage (black arrows), the presence of pulmonary exudates, and infiltration of neutrophils and mononuclear cells into the bronchial lumen (white arrows) (original magnifications, ×100 and ×200) (B and C), and bronchial necrosis (original magnification, ×200) (D). Tissue inflammation was intense at 6 dpi, with the involvement of larger portions, the presence of lymphoid structures (white arrows), and signs of emphysema (black arrow) (original magnification, ×40) (E), heavy infiltration of inflammatory cells, specifically lymphocytes, in peribronchial (white arrow) and perivascular (black arrow) areas (original magnification, ×40) (F), typical lobular pneumonia resulting in diffused alveolar damage (original magnification, ×100) (G), and signs of bronchial spasm (white arrows) (original magnification, ×100) (H).

FIG 3

Peramivir mediated protection of mice against lethal H7N9 challenge. Animals were infected with the indicated viral concentrations, and 30 mg/kg of peramivir was administered intramuscularly once daily from the time of infection until 8 dpi. Significant changes in animal body weight (a, c, and e) and lethality (b, d, and f) were observed after peramivir treatment throughout the course of infection. MDCK cells were used to titrate viral loads present in lung tissues of peramivir- or vehicle (0.85% NaCl)-treated animals at 3 dpi (g) and 6 dpi (h). Results are expressed as the log₁₀ mean TCID₅₀/ml ± standard error of the mean (SEM) for each group of mice (n = 3). *, P < 0.01; **, P < 0.001; ***, P < 0.0001.

FIG 4

Temporal changes in H7N9-induced lung pathology following peramivir treatment. The images show H&E staining of lung sections. (A) Peramivir (30 mg/kg; D0 to D8)-treated animals showed minimal resolution of lung pathology compared to untreated infected animals (D) at 6 dpi. (B) Surviving animals in the peramivir-treated group showed resolved lung pathology, as evidenced by normal lung architecture in 90% of areas and localized inflammation in certain places, at 14 dpi. Lung sections stained with influenza virus NP antibody showed minimal signs of infection in peramivir-treated animals (magnification, ×100) (C), while infection of type II pneumocytes (E), inflammatory cells, and bronchial epithelium (F) was observed in untreated infected animals (magnification, ×400).

FIG 5

Dose-dependent effect of peramivir on lethal H7N9 influenza virus challenge in mice. Animals were infected with 10⁴ EID₅₀ of A/Shantou/1001/2014 H7N9 virus and administered 30, 15, or 3 mg/kg/day of peramivir intramuscularly from 0 to 8 dpi. Peramivir provided protection against lethal H7N9 infection in a dose-dependent manner. Changes in animal weight (a) and lethality (b) were observed throughout the course of infection. (c) The AUCs for animal weights from 1 to 14 dpi showed a 2-fold improvement for peramivir-treated animals (n = 10/group). (d) Dose-dependent reductions of viral loads in lung homogenates from peramivir-treated animals were seen at 3 and 6 dpi. Results are expressed as the log₁₀ mean TCID₅₀/ml ± SEM for each group of mice (n = 3). ***, P < 0.0001; **, P < 0.001.

FIG 6

Comparison of single and multiple doses of peramivir. Animals were infected with 10⁴ EID₅₀ of A/Shantou/1001/2014 H7N9 virus. Single-dose regimens consisted of 30 mg/kg/day of peramivir administered intramuscularly at the time of infection (D0 single) or at 1 dpi (D1 single). In multiple-dose regimens, similar peramivir treatments were initiated either at the time of infection (D0 multiple) or at 1 dpi (D1 multiple) and continued until 8 dpi. Changes in animal weight (a) and lethality (b) were observed throughout the course of infection. (c) Reductions of viral loads in lung homogenates from peramivir-treated animals were seen at 3 and 6 dpi. Results are expressed as the log₁₀ mean TCID₅₀/ml ± SEM for each group of mice (n = 3). ***, P < 0.0005; **, P < 0.005.

FIG 7

Pathological changes in brain tissues after A/Shantou/1001/2014 (H7N9) infection in C57/BL6 mice. Representative sections show hemorrhaging (black arrow) (A), degenerative neurons and infiltrating cells (B), liquefaction of neural cells in the midbrain (C and D), an increased size of the arachnoid space (E), and hemorrhaging in the cerebral cortex (E and F). (G and H) Infiltration of inflammatory cells (black arrow) and neural edema (white arrow) in the frontal cortex at 3 dpi. (I and J) Karyopyknosis (black arrow) in the cerebrum at 6 dpi. (K and L) Minimal signs of hemorrhage associated with neural degeneration in H7N9-infected animals after treatment with 30 mg/kg of peramivir at 3 dpi. Left panels show higher-magnification (×400) views of the boxes in the right panels.