Association of increased pathogenicity of Asian H5N1 highly pathogenic avian influenza viruses in chickens with highly efficient viral replication accompanied by early destruction of innate immune responses

Abstract

The Asian H5N1 highly pathogenic avian influenza (HPAI) viruses have been increasing in pathogenicity in diverse avian species since 1996 and are now widespread in Asian, European, and African countries. To better understand the basis of the increased pathogenicity of recent Asian H5N1 HPAI viruses in chickens, we compared the fevers and mean death times (MDTs) of chickens infected with the Asian H5N1 A/chicken/Yamaguchi/7/04 (CkYM7) strain with those infected with the H5N1 Duck/Yokohama/aq10/03 (DkYK10) strain, using a wireless thermosensor. Asian H5N1 CkYM7 caused peracute death in chickens before fever could be induced, whereas DkYK10 virus induced high fevers and had a long MDT. Real-time PCR analyses of cytokine mRNA expressions showed that CkYM7 quickly induced antiviral and proinflammatory cytokine mRNA expressions at 24 h postinfection (hpi) that suddenly decreased at 32 hpi. In contrast, these cytokine mRNA expressions increased at 24 hpi in the DkYK10 group, but decreased from 48 hpi onward to levels similar to those resulting from infection with the low-pathogenicity H5N2 A/chicken/Ibaraki/1/2004 strain. Sequential titrations of viruses in lungs, spleens, and kidneys demonstrated that CkYM7 replicated rapidly and efficiently in infected chickens and that the viral titers were more than twofold higher than those of DkYK10. CkYM7 preferentially and efficiently replicated in macrophages and vascular endothelial cells, while DkYK10 grew moderately in macrophages. These results indicate that the increased pathogenicity in chickens of the recent Asian H5N1 HPAI viruses may be associated with extremely rapid and high replication of the virus in macrophages and vascular endothelial cells, which resulted in disruption of the thermoregulation system and innate immune responses.

FIG. 1.

Survival of 4-week-old chickens after intranasal inoculation with one of two HPAI viruses or an LPAI virus. Shown is the percent survival of chickens inoculated with CkYM7 (H5N1), DkYK10 (H5N1), or CkIB1 (H5N2) or mock inoculated. The remaining chickens were observed for clinical signs and gross lesions twice a day through a 10-day observation period.

FIG. 2.

Body temperature kinetics of chickens after intranasal inoculation with one of two HPAI viruses or an LPAI virus. The average kinetics are shown. Each curve shows the mean ± standard deviation for 7 to 10 chickens. (A) CkYM7 (H5N1); (B) DkYK10 (H5N1); (C) CkIB1 (H5N2); and (D) mock.

FIG. 3.

Fever and MDT of chickens after intranasal inoculation. Values represent the mean ± standard deviation for each group. (A) Fever is based on increases above the basal body temperature. Average fever per group is shown. (B) The time to death of each chicken was defined as the time when body temperature fell below 30°C after inoculation; the MDT for each group is shown. An asterisk denotes significant difference from the value for CkYM7 (H5N1) (P < 0.05).

FIG. 4.

Effect of virus dose on fever and MDT of chickens infected with CkYM7, DkYK10, or CkIB1. Four-week-old SPF White Leghorn chickens (four chickens per dilution) were intranasally inoculated with 0.1 ml of diluted virus. MDT and average fevers of the chickens after inoculation with CkYM7 (A and B) and DkYK10 (C and D) are shown. Values represent the mean ± standard deviation for each dilution.

FIG. 5.

Kinetic curves of mRNA expression of IFN-α, IFN-β, IFN-γ, IL-4, IL-6, IL-8, IL-10, IL-15, and IL-18 genes in the lungs of chickens infected with AI viruses. The data are the mean change (fold) ± standard deviation for cytokine mRNA expression levels in each group. In contrast with CkIB1, CkYM7 and DkYK10 induced higher levels of IFN-α, IFN-β, IL-6, IL-8, IL-15, and IL-18.

FIG. 6.

Kinetics of virus titers in chicken tissues. Titers of individual lungs, kidneys, and spleens were determined with embryonated chicken eggs. The mean virus titers ± standard deviations from three birds are shown (log₁₀ EID₅₀/g).

FIG. 7.

Immunohistochemical staining of AI virus matrix protein in lungs and spleens of chickens intranasally inoculated with CkYM7 or DkYK10. The viral M antigen was detected mainly in vascular endothelial cells and macrophages of lung tissue 32 h after inoculation with CkYM7 (A), macrophages of lung tissue 81 h after inoculation with DkYK10 (B), and macrophages in splenic red pulp 32 h after inoculation with CkYM7 (C). (D) DkYK10 in spleen at 81 hpi. Magnification: ×40 (A to D).

FIG. 8.

Detection of apoptotic cells in lungs and spleens of chickens infected with CkYM7 or DkYK10. The apoptotic cells were detected in lung (A) and spleen (D) tissues 36 h after inoculation with CkYM7 and lung (B) and spleen (E) tissues 96 h after inoculation with DkYK10. Lung (C) and spleen (F) tissues derived from uninfected chickens. Magnification: ×40 (A to F).