NP, PB1, and PB2 viral genes contribute to altered replication of H5N1 avian influenza viruses in chickens

Abstract

The virulence determinants for highly pathogenic avian influenza viruses (AIVs) are considered multigenic, although the best characterized virulence factor is the hemagglutinin (HA) cleavage site. The capability of influenza viruses to reassort gene segments is one potential way for new viruses to emerge with different virulence characteristics. To evaluate the role of other gene segments in virulence, we used reverse genetics to generate two H5N1 recombinant viruses with differing pathogenicity in chickens. Single-gene reassortants were used to determine which viral genes contribute to the altered virulence. Exchange of the PB1, PB2, and NP genes impacted replication of the reassortant viruses while also affecting the expression of specific host genes. Disruption of the parental virus' functional polymerase complexes by exchanging PB1 or PB2 genes decreased viral replication in tissues and consequently the pathogenicity of the viruses. In contrast, exchanging the NP gene greatly increased viral replication and expanded tissue tropism, thus resulting in decreased mean death times. Infection with the NP reassortant virus also resulted in the upregulation of gamma interferon and inducible nitric oxide synthase gene expression. In addition to the impact of PB1, PB2, and NP on viral replication, the HA, NS, and M genes also contributed to the pathogenesis of the reassortant viruses. While the pathogenesis of AIVs in chickens is clearly dependent on the interaction of multiple gene products, we have shown that single-gene reassortment events are sufficient to alter the virulence of AIVs in chickens.

FIG. 1.

Survival after IN inoculation with the reassortant viruses. Two-week-old chickens were inoculated with 0.1 ml of an inoculum containing 10⁶ EID₅₀/ml of the reassortant viruses, and mortality was monitored for 10 days. Parent virus and control survival analysis (A) and reassortant virus survival analysis (B). All groups are statistically different from rEgret and rIndo/rEgret NP (P < 0.05) as determined by the log rank test. Explanation of footnotes in figure key: a, survival is significantly different from the control group and also from the rEgret group; b, survival is significantly different from all groups and the control group; c, survival is significantly different from the control group but not the rIndo group; d, survival is not significantly different from the control group or the rIndo group; e, survival is significantly different from the rIndo group but not the control group.

FIG. 2.

Experimental studies of chickens that were IN inoculated with AIV H5N1 recombinant viruses; representative microscopic lesions and immunohistologic findings. Photomicrographs (×200) of tissue sections stained with hematoxylin and eosin (A, C, E, and G) or by immunohistochemistry to demonstrate AIV (B, D, F, H, I, and J). (A) Histiocytic interstitial pneumonia in a 2-week-old chicken inoculated with rEgret, 2 dpi, with congestion and fibrous exudates in the airways. (B) AI viral antigen (red, arrows) in macrophages and air capillary and blood vessel endothelium in lung tissue of the same chicken. (C) Spleen tissue of a 2-week-old chicken inoculated with rEgret, 2 dpi; mild focal splenitis. (D) AI viral antigen (red, arrow) in macrophages and vascular endothelial cells in spleen tissue of the same chicken. (E) Severe interstitial pneumonia in a 2-week-old chicken inoculated with rIndo/Egret NP, 2 dpi. There is congestion, mononuclear infiltrates, and serofibrinous exudates filling air capillaries. (F) Diffuse staining (red) for AI viral antigen in the endothelium and infiltrating macrophages in lung tissue of the same chicken. (G) Splenic necrosis in a 2-week-old chicken inoculated with rIndo/Egret NP, 2 dpi. (H) AI viral antigen (red) in macrophages in spleen tissue of the same chicken. (I) AI viral antigen (red) in brain tissue of a 2-week-old chicken inoculated with rIndo/Egret NP, 2 dpi. Staining present in neurons and glial cells of brain tissue. (J) AI viral antigen (red) in the myocardial cells of the heart of a 2-week-old chicken inoculated with rIndo/Egret NP, 2 dpi.

FIG. 3.

Virus titers in spleen, lung, and brain tissues. Tissues were taken 2 dpi and homogenized to a 10% (wt/vol) final concentration in BHI medium. A portion (100 μl) of 10-fold dilutions of the homogenates was inoculated into 10-day-old ECEs, and log₁₀ EID₅₀/g was calculated. Values are the mean ± standard error (n = 2) with the exception of the rIndo/rEgret PB1 lung titer (n = 1). The threshold of detection is 2.4 log₁₀ EID₅₀/gram of tissue.

FIG. 4.

Semiquantitative analysis of differential mRNA gene expression in the spleen tissue of chickens infected with AIV recombinants. Total cellular RNA was extracted from spleen tissue collected 2 dpi from three chickens. Equal amounts of RNA from the three chickens per group were pooled prior to RT-PCR analysis. Analysis of the pooled RNA was carried out using different primer sets with β-actin as an amplification and loading control (n = 1). Bands were quantified, and intensities shown were normalized to the β-actin control. Gene expression of control birds was arbitrarily set to 1.

FIG. 5.

Semiquantitative analysis of differential mRNA gene expression in lung tissue from chickens infected with AIV recombinants. Total cellular RNA was extracted from spleen and lung tissues from three chickens 2 dpi. Equal amounts of RNA from the three chickens per group were pooled prior to RT-PCR analysis. Analysis was carried out using different primer sets with β-actin as an amplification and loading control. Bands were quantified, and intensities shown were normalized to the β-actin control. Gene expression of control birds was arbitrarily set to 1.