Avian influenza (H5N1) viruses isolated from humans in Asia in 2004 exhibit increased virulence in mammals

Abstract

The spread of highly pathogenic avian influenza H5N1 viruses across Asia in 2003 and 2004 devastated domestic poultry populations and resulted in the largest and most lethal H5N1 virus outbreak in humans to date. To better understand the potential of H5N1 viruses isolated during this epizootic event to cause disease in mammals, we used the mouse and ferret models to evaluate the relative virulence of selected 2003 and 2004 H5N1 viruses representing multiple genetic and geographical groups and compared them to earlier H5N1 strains isolated from humans. Four of five human isolates tested were highly lethal for both mice and ferrets and exhibited a substantially greater level of virulence in ferrets than other H5N1 viruses isolated from humans since 1997. One human isolate and all four avian isolates tested were found to be of low virulence in either animal. The highly virulent viruses replicated to high titers in the mouse and ferret respiratory tracts and spread to multiple organs, including the brain. Rapid disease progression and high lethality rates in ferrets distinguished the highly virulent 2004 H5N1 viruses from the 1997 H5N1 viruses. A pair of viruses isolated from the same patient differed by eight amino acids, including a Lys/Glu disparity at 627 of PB2, previously identified as an H5N1 virulence factor in mice. The virus possessing Glu at 627 of PB2 exhibited only a modest decrease in virulence in mice and was highly virulent in ferrets, indicating that for this virus pair, the K627E PB2 difference did not have a prevailing effect on virulence in mice or ferrets. Our results demonstrate the general equivalence of mouse and ferret models for assessment of the virulence of 2003 and 2004 H5N1 viruses. However, the apparent enhancement of virulence of these viruses in humans in 2004 was better reflected in the ferret.

FIG. 1.

Phylogenetic relationships of the H5 hemagglutinin genes. The tree includes avian influenza isolates collected during the 2003 and 2004 outbreak in Asia and selected ancestors dating back to 1996 (see Materials and Methods for database accession numbers). The viruses evaluated in this study are in boldface type. HA clades are indicated by curved brackets. Phylogenetic trees were inferred from nucleotide sequences by the neighbor-joining method with A/Chicken/Scotland/56 genes as the outgroup (not shown; the branch position is indicated by the arrow). Bootstrap analysis values of ≥90% are shown above the branches. The scale bar indicates the number of nucleotide (nt) changes per unit length of the horizontal branches.

FIG. 2.

Comparison of mean titers of influenza A H5N1 virus recovered from mouse tissues. Mice were inoculated i.n. with 10⁶ EID₅₀ of each virus, and tissues were collected on days 3 (A) and 6 (B) p.i. Tissue homogenates were prepared and titrated in eggs. Virus endpoint titers are expressed as mean log₁₀ EID₅₀ per milliliter plus standard deviation. The limit of virus detection was 10^1.5 EID₅₀/ml.

FIG. 3.

Weight change of H5N1-infected ferrets. All ferrets were inoculated i.n. with 10⁷ EID₅₀ of H5N1 virus. The percent weight change was determined by comparing the weight of each animal at each time point to its preinfection weight. The mean percentage weight change is shown ± standard deviation. (A) Ferrets were inoculated with either VN1203 (⧫), VN1204 (▪), Thai16 (▴), or Kan353 (×) and were weighed on days −1, 0, 1, 3, 5, 7, and 9 p.i. Only a single ferret remained on day 7 p.i. for Thai16 and on day 9 p.i. for VN1204 and Kan353. (B) Ferrets were inoculated with either SP83 (⧫), CkIndon (▪), CkKorea (▴), or CkNCVD31 (×) and were weighed on days −1, 0, 1, 3, 5, and 7 p.i.

FIG. 4.

Lethality and neurological dysfunction exhibited by ferrets infected with H5N1 viruses. Infected ferrets were monitored for a 14-day experimental period for clinical signs of illness. Any ferret that exhibited neurological signs or lost more than 25% of its body weight was euthanatized. Each bar indicates the percentage of ferrets that died or were euthanatized before the end of the experimental period, while the dark shaded portion of the bar indicates the portion of those ferrets exhibiting neurological dysfunction. Data from ferrets infected with A/Duck/Anyang/AVL-1/01 (24) or A/Hong Kong/483/97 virus (41) were published elsewhere. The percentage for A/Hong Kong/486/1997 virus was determined based on data obtained here and from Zitzow et al. (41).

FIG. 5.

Virus replication in the upper respiratory tract of H5N1-infected ferrets. Virus titers were measured in nasal washes collected on the days indicated from ferrets inoculated i.n. with 10⁷ EID₅₀ of either an H5N1 virus characterized as highly virulent (A) or an H5N1 virus found to be of low virulence (B). Mean titers are shown and are expressed as the log₁₀ mean (plus standard deviation) EID₅₀/ml, with the limit of detection at 10^1.5 EID₅₀/ml.

FIG. 6.

Systemic replication of H5N1 viruses in ferrets. Virus titers in tissues collected 3 days p.i. from ferrets infected with 10⁷ EID₅₀ of the viruses indicated were measured in eggs. Mean viral titers are shown and are expressed as EID₅₀/g plus standard deviation for all tissues except the nasal turbinates, which are expressed as EID₅₀/ml. The limit of detection was 10^1.5 EID₅₀/ml of tissue homogenate. ND, not determined.

FIG. 7.

Representative images of histopathologic changes and immunostaining of tissues from ferrets infected with H5N1 viruses of high or low virulence. (A) H&E staining of the lungs of a VN1204-infected ferret 3 days p.i. showing diffuse interstitial inflammation accompanied by intra-alveolar edema. (B) H&E staining of the lungs of an SP83-infected ferret 3 days p.i. showing more modest inflammation and more gas-filled lumina than in those infected with a highly virulent virus. (C) Immunostaining of influenza virus antigen in the lungs of a VN1204-infected ferret 3 days p.i. was observed in bronchiolar epithelial cells and interstitial cells (inset). (D) H&E staining of the parietal cortex of the brain from a VN1204-infected ferret 9 days p.i. showing inflammatory infiltrate in the meninges and brain parenchyma. (E) H&E staining of the parietal cortex of the brain tissue from a ferret infected with SP83 virus 14 days p.i. showing a lack of inflammatory infiltrate. (F) Immunostaining of brain tissue from a VN1204-infected ferret 9 days p.i. exhibiting influenza virus antigen in neurons in an area of brain inflammation. Original magnifications, ×50 (A, B, D, and E); ×157.5 (C); ×100 (F).