Role of poultry in the spread of novel H7N9 influenza virus in China

Abstract

The recent outbreak of H7N9 influenza in China has resulted in many human cases with a high fatality rate. Poultry are the likely source of infection for humans on the basis of sequence analysis and virus isolations from live bird markets, but it is not clear which species of birds are most likely to be infected and shedding levels of virus sufficient to infect humans. Intranasal inoculation of chickens, Japanese quail, pigeons, Pekin ducks, Mallard ducks, Muscovy ducks, and Embden geese with 10(6) 50% egg infective doses of the A/Anhui/1/2013 virus resulted in infection but no clinical disease signs. Virus shedding was much higher and prolonged in quail and chickens than in the other species. Quail effectively transmitted the virus to direct contacts, but pigeons and Pekin ducks did not. In all species, virus was detected at much higher titers from oropharyngeal swabs than cloacal swabs. The hemagglutinin gene from samples collected from selected experimentally infected birds was sequenced, and three amino acid differences were commonly observed when the sequence was compared to the sequence of A/Anhui/1/2013: N123D, N149D, and L217Q. Leucine at position 217 is highly conserved for human isolates and is associated with α2,6-sialic acid binding. Different amino acid combinations were observed, suggesting that the inoculum had viral subpopulations that were selected after passage in birds. These experimental studies corroborate the finding that certain poultry species are reservoirs of the H7N9 influenza virus and that the virus is highly tropic for the upper respiratory tract, so testing of bird species should preferentially be conducted with oropharyngeal swabs for the best sensitivity.

Importance: The recent outbreak of H7N9 influenza in China has resulted in a number of human infections with a high case fatality rate. The source of the viral outbreak is suspected to be poultry, but definitive data on the source of the infection are not available. This study provides experimental data to show that quail and chickens are susceptible to infection, shed large amounts of virus, and are likely important in the spread of the virus to humans. Other poultry species can be infected and shed virus but are less likely to play a role of transmitting the virus to humans. Pigeons were previously suggested to be a possible source of the virus because of isolation of the virus from several pigeons in poultry markets in China, but experimental studies show that they are generally resistant to infection and are unlikely to play a role in the spread of the virus.

FIG 1

Comparison of oropharyngeal virus shedding after experimental challenge. Oropharyngeal shedding was detected at 2, 4, 6, 8, and 11 dpi with 10⁶ EID₅₀ of A/Anhui/1/2013 (H7N9) virus. The rRT-PCR values were interpolated by quantitative real-time RT-PCR using a standard curve generated with the challenge isolate. The estimated lower limit of sensitivity of the rRT-PCR test was 10², as determined by the limit of detection on the standard curve. The results are presented as the average viral shedding for each day, and samples negative by rRT-PCR were given a value 1 log unit lower than the limit of detection.

FIG 2

Histopathology and immunohistochemical staining for avian influenza virus antigen in tissues of quail intranasally infected with the A/Anhui/1/2013 (H7N9) virus 3 dpi. Virus is stained in red. (A and B) Nasal epithelium. Severe necrotizing rhinitis with submucosal congestion and edema, glandular hyperplasia, and lymphoplasmacytic infiltration (A) and viral antigen in epithelial cells (B) are shown (magnification, ×200). (C and D) Nasal epithelium. Necrosis of epithelial cells and lymphocytic infiltration (arrowhead) (C) and viral antigen in epithelial cells (D) are shown (magnification, ×400). (E and F) Nasal gland. Lymphocytic infiltration in submucosa (arrowheads) (E) and viral antigen in epithelial cells (F) are shown (magnification, ×200). (G and H) Trachea. Necrosis of epithelial cells (arrowhead) (G) and viral antigen staining in epithelial cells (H) are shown (magnification, ×400).