Pathogenesis of avian influenza A (H5N1) viruses in ferrets

Abstract

Highly pathogenic avian influenza A H5N1 viruses caused outbreaks of disease in domestic poultry and humans in Hong Kong in 1997. Direct transmission of the H5N1 viruses from birds to humans resulted in 18 documented cases of respiratory illness, including six deaths. Here we evaluated two of the avian H5N1 viruses isolated from humans for their ability to replicate and cause disease in outbred ferrets. A/Hong Kong/483/97 virus was isolated from a fatal case and was highly pathogenic in the BALB/c mouse model, whereas A/Hong Kong/486/97 virus was isolated from a case with mild illness and exhibited a low-pathogenicity phenotype in mice. Ferrets infected intranasally with 10(7) 50% egg infectious doses (EID(50)) of either H5N1 virus exhibited severe lethargy, fever, weight loss, transient lymphopenia, and replication in the upper and lower respiratory tract, as well as multiple systemic organs, including the brain. Gastrointestinal symptoms were seen in some animals. In contrast, weight loss and severe lethargy were not noted in ferrets infected with 10(7) EID(50) of two recent human H3N2 viruses, although these viruses were also isolated from the brains, but not other extrapulmonary organs, of infected animals. The results demonstrate that both H5N1 viruses were highly virulent in the outbred ferret model, unlike the differential pathogenicity documented in inbred BALB/c mice. We propose the ferret as an alternative model system for the study of these highly pathogenic avian viruses.

FIG. 1.

Kinetics of virus replication in the upper respiratory tract and weight loss in H5N1-infected ferrets. Nine ferrets were inoculated i.n. with 10⁷ EID₅₀ of either HK/483 (▪) or HK/486 (▴). The number of animals assessed on different days was as follows: days 0 and 1, n = 9; day 3, n = 7; day 5, n = 5; and days 7, 9 and 11, n = 3. (A) Nasal wash samples were collected from ferrets on the days indicated; virus titers were determined in eggs and are expressed as the log₁₀ mean ± SE EID₅₀/ml. The limit of virus detection was ≤10^1.0/ml. (B) Weights of animals were recorded on the days indicated, and the individual differences in weight compared with weights prior to infection were calculated for each individual animal. The mean percent change in weight is presented. Weight loss in HK/483-infected ferrets was greater than in HK/486-infected ferrets on day 11 p.i. (P = 0.002). The change in weight in six mock-infected control animals ranged from −1.4 to +3.9%.

FIG. 2.

Change in temperatures of ferrets infected with H5N1 viruses. Ferrets were infected with 10⁷ EID₅₀ of either HK/483 or HK/486. Temperatures were assessed twice daily, beginning 3 days prior to infection and for 14 days after infection. The numbers of animals monitored each day were as follows: days −3 to 1, n = 9; days 2 and 3, n = 7; days 4 and 5, n = 5; and days 7 to 14, n = 3. The means of preinfection temperatures (days −3 to −1) were subtracted from the individual p.i. temperatures to obtain temperature changes in individual ferrets. The mean change in temperature for all ferrets in a group is shown. Three ferrets inoculated with each virus were sampled on remaining days. The mean temperature change in six mock-infected control ferrets ranged from −0.5 to +0.4°C.

FIG. 3.

Replication of H5N1 viruses in ferret tissues. Ferrets were infected with 10⁷ EID₅₀ of either HK/483 or HK/486. Tissues were harvested from two to four animals on the indicated days and titers were determined in eggs. For solid tissues, viral titers are expressed as log₁₀ EID₅₀/g, and for nasal turbinates, titers are expressed as log₁₀ EID₅₀/ml. The limit of virus detection was ≤10^1.0/ml for nasal turbinates and ≤10^1.0/g for all other tissues.

FIG. 4.

Representative histolopathologic changes and immunostaining in tissues from ferrets infected with 10⁷ EID₅₀ of H5N1 or H3N2 virus. Tissues were removed at the indicated times p.i. and were processed for H&E and immunohistochemical staining. (A) H&E staining of the lungs of an HK/486-infected animal on day 3 p.i. showing extensive bronchiolar inflammation, necrosis of bronchial epithelium, and suppurative exudates in the bronchiolar lumen. (B) Immunostaining of influenza virus in lung of an HK/486-infected animal on day 3 p.i. Immunoalkaline phosphate staining, naphthol fast red substrate with light hematoxylin counterstain. (C) H&E staining of lung collected on day 3 p.i. from mock-infected control ferret showing normal histology. (D) H&E staining of lung of H3N2 (Panama/99)-infected ferret on day 3 p.i. showing interstitial pneumonitis. (E) H&E staining of brain of an HK/486-infected animal on day 5 p.i. showing presence of glial nodules. (F) H&E staining of brain of an HK/483-infected animal on day 14 p.i. showing presence of glial nodules. (G) H&E staining of brain of an HK/486-infected animal on day 14 p.i. showing prominent perivascular infiltrate. (H) H&E staining of brain of an HK/486-infected animal that died on day 9 p.i. showing neuronophagia. Magnifications: ×48 (A), ×48 (B), ×48 (C), ×48 (D), ×96 (E), ×96 (F), ×48 (G), ×96 (H).