Continued circulation in China of highly pathogenic avian influenza viruses encoding the hemagglutinin gene associated with the 1997 H5N1 outbreak in poultry and humans

Abstract

Since the outbreak in humans of an H5N1 avian influenza virus in Hong Kong in 1997, poultry entering the live-bird markets of Hong Kong have been closely monitored for infection with avian influenza. In March 1999, this monitoring system detected geese that were serologically positive for H5N1 avian influenza virus, but the birds were marketed before they could be sampled for virus. However, viral isolates were obtained by swabbing the cages that housed the geese. These samples, known collectively as A/Environment/Hong Kong/437/99 (A/Env/HK/437/99), contained four viral isolates, which were compared to the 1997 H5N1 Hong Kong isolates. Analysis of A/Env/HK/437/99 viruses revealed that the four isolates are nearly identical genetically and are most closely related to A/Goose/Guangdong/1/96. These isolates and the 1997 H5N1 Hong Kong viruses encode common hemagglutinin (H5) genes that have identical hemagglutinin cleavage sites. Thus, the pathogenicity of the A/Env/HK/437/99 viruses was compared in chickens and in mice to evaluate the potential for disease outbreaks in poultry and humans. The A/Env/HK/437/99 isolates were highly pathogenic in chickens but caused a longer mean death time and had altered cell tropism compared to A/Hong Kong/156/97 (A/HK/156/97). Like A/HK/156/97, the A/Env/HK/437/99 viruses replicated in mice and remained localized to the respiratory tract. However, the A/Env/HK/437/99 isolates caused only mild pathological lesions in these tissues and no clinical signs of disease or death. As a measure of the immune response to these viruses, transforming growth factor beta levels were determined in the serum of infected mice and showed elevated levels for the A/Env/HK/437/99 viruses compared to the A/HK/156/97 viruses. This study is the first to characterize the A/Env/HK/437/99 viruses in both avian and mammalian species, evaluating the H5 gene from the 1997 Hong Kong H5N1 isolates in a different genetic background. Our findings reveal that at least one of the avian influenza virus genes encoded by the 1997 H5N1 Hong Kong viruses continues to circulate in mainland China and that this gene is important for pathogenesis in chickens but is not the sole determinant of pathogenicity in mice. There is evidence that H9N2 viruses, which have internal genes in common with the 1997 H5N1 Hong Kong isolates, are still circulating in Hong Kong and China as well, providing a heterogeneous gene pool for viral reassortment. The implications of these findings for the potential for human disease are discussed.

FIG. 1

Phylogenetic analysis of the nucleotide sequence of the HA and M genes of the A/Env/HK/437/99 isolates. Trees were generated using parsimony with a heuristic search method by bootstrap analysis with 100 replicates and PAUP 3.1 (37). Branch lengths give the number of nucleotide changes, and trees are midpoint rooted. All isolates are type A influenza viruses. Abbreviations: CK, chicken; DK, duck; Env, environment; FPV, fowl plague virus; GS, goose; TK, turkey; standard two-letter abbreviations are used for states in the United States. (A) HA1 subunit of the H5 HA gene segment. (B) M gene segment.

FIG. 2

Weights of mice that were mock infected or infected with A/Env/HK/437-6/99, A/Env/HK/437-10/99, or A/HK/156/97. The weights were determined on days 0, 4, 6, 8, and 12 postinoculation. The weights of the mice were averaged for each group on the days indicated, and error bars show the standard deviation of the mean.

FIG. 3

Experimental studies in 4-week-old chickens and 7-week-old mice inoculated with A/Env/HK/437-4/99, A/Env/HK/437-6/99, A/Env/HK/437-8/99, and A/Env/HK/437-10/99. Photomicrographs of hematoxylin and eosin-stained tissue sections (a, b, f, and g) and photomicrographs of tissue sections stained immunohistochemically to demonstrate avian influenza virus NP (c to e and h) are shown. (a) Neuronal degeneration and necrosis in the medulla of a chicken euthanized on day 2 after intravenous inoculation with A/Env/HK/437-6/99. Bar, 25 μm. (b) Severe widespread necrosis of pancreatic acinar epithelium from a chicken euthanized on day 2 after intravenous inoculation with A/Env/HK/437-8/99. Bar, 20 μm. (c) Intranuclear and intracytoplasmic avian influenza virus antigen in neurons and glial cells from the chicken in panel a. Bar, 50 μm. (d) Intense staining of pancreatic acinar epithelium and debris for avian influenza virus antigen from the chicken in panel b. Bar, 50 μm. (e) Intranuclear and intracytoplasmic avian influenza virus antigen in cardiac myocytes of a chicken that died on day 2 after intravenous inoculation with A/Env/HK/437-10/99. Bar, 50 μm. (f) Single focal area of bronchitis in a normal lung from a mouse euthanized on day 4 after intranasal inoculation with A/Env/HK/437-4/99. Bar, 500 μm. (g) Higher magnification of panel f, showing focal necrosis of respiratory epithelium from a bronchus. Bar, 50 μm. (h) Avian influenza virus antigen in bronchial respiratory epithelium of the mouse in panel f. Bar, 50 μm.

FIG. 4

TGF-β activity levels in the serum of mock- and virus-infected mice. TGF-β activity levels were determined using an NRK soft-agar assay and are reported in number of colonies formed. A standard curve was derived from known levels of TGF-β. EGF alone represents the background level of colony formation in this assay. Mice were mock infected or infected with A/Env/HK/437-6/99, A/Env/HK/437-10/99, or A/HK/156/97, and serum was evaluated on days 1 and 4 postinoculation (dpi). Numbers above the experimental samples represent the amount (in picograms) of activated TGF-β per 50 μl of serum as determined using the standard curve generated from known amounts of TGF-β. The value could not be accurately estimated for mock-infected or A/HK/156/97-infected mouse serum because the number of colonies formed was smaller than that of the lowest TGF-β value determined for the standard curve. Experiments were performed in triplicate and are represented as the mean of the samples and the standard error of the mean.