Experimental infection of macaques with a wild water bird-derived highly pathogenic avian influenza virus (H5N1)

Abstract

Highly pathogenic avian influenza virus (HPAIV) continues to threaten human health. Non-human primate infection models of human influenza are desired. To establish an animal infection model with more natural transmission and to determine the pathogenicity of HPAIV isolated from a wild water bird in primates, we administered a Japanese isolate of HPAIV (A/whooper swan/Hokkaido/1/2008, H5N1 clade 2.3.2.1) to rhesus and cynomolgus monkeys, in droplet form, via the intratracheal route. Infection of the lower and upper respiratory tracts and viral shedding were observed in both macaques. Inoculation of rhesus monkeys with higher doses of the isolate resulted in stronger clinical symptoms of influenza. Our results demonstrate that HPAIV isolated from a water bird in Japan is pathogenic in monkeys by experimental inoculation, and provide a new method for HPAIV infection of non-human primate hosts, a good animal model for investigation of HPAIV pathogenicity.

Figure 1. Activity of cynomolgus and rhesus monkeys challenged with HPAIV.

The activity of each monkey challenged with 3 × 10⁶ PFU of HPAIV was monitored by a telemetry system. Amplitude indicates activity of an animal. *Period with decreased activity; AUC was decreased to ≤ 50% of the pre-inoculation level. #Data not obtained. The arrow indicates the time of anesthesia for sampling. Hyperthermia was caused by anesthesia.

Figure 2. Changes in respiratory rate in HPAIV-challenged monkeys.

The respiratory rate was measured soon after anesthesia, daily (cynomolgus monkeys) or at 0, 2, 4, 6, and 7 dpi (rhesus monkeys).

Figure 3. Histopathology of the lungs of cynomolgus and rhesus macaques challenged with HPAIV.

(A) Histopathology of the lung lobes was scored as: 0, none; 1, mild (area of inflammation was ≤1/3 of the observed section); 2, intermediate (area of inflammation was between 1/3 and 2/3 of the observed section); 3, severe (area of inflammation was ≥2/3 of the observed section). * Not analyzed. (B) Hematoxylin and eosin stains of the right upper lobe of the lungs of rhesus monkey 31 (×40). (C) Inset of (B) is magnified (×400).

Figure 4. The amount of viral RNA in the respiratory tract between cynomolgus and rhesus macaques.

Viral RNA was quantified by qRT-PCR. The log value of the relative viral RNA content is indicated.

Figure 5. Numbers of leukocytes and lymphocytes in blood of HPAIV-infected macaques.

Blood was collected daily from cynomolgus (A) or rhesus monkeys (B) at 0, 2, 4, 6, and 7 dpi from rhesus monkeys. The number of lymphocytes was calculated from the number of total leukocytes and the lymphocyte ratio.

Figure 6. The amounts of viral RNA in the respiratory tract in rhesus and cynomolgus monkeys between different inoculation methods.

(A) A rhesus monkey was inoculated with HPAIV via a catheter. Viral RNA in the respiratory tract (two parts for the upper respiratory tract, three parts for the trachea, two parts for the bronchus, and six parts for the lungs) was quantified by qRT-PCR. The total amount of viral RNA in each organ is indicated as the log value. Data on the three rhesus monkeys (#29−31) shown in Figure 4 was similarly analyzed and the mean±SEM for three monkeys is indicated. (B) Cynomolgus monkeys were inoculated with HPAIV via liquid exposure (n=2) or droplet exposure (n=1). Data were analyzed similarly to (A), including data for cynomolgus monkeys (#23−25). Data are means±SD was indicated.

Figure 7. Body temperature and activity in rhesus monkeys challenged with higher infectious doses of HPAIV.

Body temperature (A) and activity (B) of rhesus monkeys were monitored. The onset of fever was observed (arrowheads). *Period with decreased activity; AUC was decreased to ≤ 50% of the pre-inoculation level. The time points of anesthesia are indicated by arrows. Hyperthermia was caused by anesthesia.

Figure 8. Clinical symptoms in rhesus monkeys challenged with HPAIV.

Respiratory rate (A) and body weight (B) were measured soon after anesthesia. The amount of consumed food was recorded daily (C). Orange and red lines show the data of the monkeys inoculated with 1 × 10⁷ PFU (#27, circle; #28, square) or 1 × 10⁸ PFU (#32, circle; #33, square), respectively. A purple line shows the mean of the data of the monkeys inoculated with 3 × 10⁶ PFU.

Figure 9. Progression of pneumonia in rhesus macaques after HPAI infection.

(A) Radiographic photographs of the chest were taken every other day in rhesus macaques. (B). Histopathology of the lung lobes was scored as: 0, none; 1, mild; 2, severe. * Not analyzed.

Figure 10. The amount of viral RNA in the respiratory tract among rhesus monkeys inoculated with different doses of HPAIV.

Viral RNA was quantified by qRT-PCR. The log value of relative viral RNA content is indicated.

Figure 11. The numbers of lymphocytes after HPAIV infection.

The number of lymphocyte was examined as described in Figure 5.