Phylogenetic and molecular characterization of H9N2 influenza isolates from chickens in Northern China from 2007-2009

Abstract

The repeated transmission to pigs and humans, and the long-term endemicity in terrestrial poultry of H9N2 viruses in China lend urgency to the study of their ecology and pathogenicity. In the present paper, we reported an H9N2 virus sublineage isolated from chickens in northern China from 2007 to 2009 has high lethality for mice. Phylogenetic analysis of the full genome indicated that six representative H9N2 isolates shared high homology to each other, and they clustered in the same sublineage with other H9N2 viruses isolated recently in northern China. The isolates were double-reassortant viruses containing M genes similar to A/Quail/Hong Kong/G1/97 (H9N2) and the other seven gene segments from A/Chicken/Shanghai/F/98 (H9N2). These six isolates were capable of replicating in the lungs of infected chickens without producing observable clinical signs of disease or death. However, they were highly lethal to mice with mortality rates as high as 100% (14/14) without prior adaptation. The affected mice exhibited severe respiratory syndromes and diffuse lung injury. The H9N2 viruses could be detected in multiple organs of the infected mice, including hearts, livers, spleens, lungs and kidneys. Our findings demonstrated that H9N2 viruses isolated from the chickens in northern China have established a stable sublineage with enhanced pathogenicity to mice, suggesting that urgent attention will need to be paid to the transmission of H9N2 viruses from chickens to mammals.

Figure 1. Phylogenetic trees for HA (A), NA (B), M (C) and NS (D) genes of the H9N2 influenza viruses analyzed.

Trees were generated by the neighbor-joining method with the MEGA program (version 4.1). Nucleotides 80–1090 (1011 bp, HA), 20–1420 (1401 bp, NA), 26–984 (959 bp, M) and 41–859 (819 bp, NS) were used for phylogenetic analysis. The HA phylogenetic tree is rooted to A/Duck/Alberta/60/76(H12N5), and the trees of NA, M, and NS are rooted to A/Equine/Praque/1/56(H7N7). The length of the horizontal lines is proportional to the minimum number of nucleotide differences required to join nodes. Vertical lines are for spacing and labeling. Viruses characterized in this study are highlighted in bold, representative H9N2 viruses are in red, viruses isolated from swine and human are in green, and H5N1 influenza viruses are highlighted with asterisk. Abbreviations: Ck, chicken; Dk, duck; Sw, swine; Gf, guinea fowl; Pg, pigeon; Gs, goose; Qa, quail; Ty, turkey; Pf, peregrine falcon; WDk, wild duck; Cu, chukar; HK, Hong Kong; WI, Wisconsin, Bei, Beijing; CA, California; VNM; Viet Nam.

Figure 2. Phylogenetic trees for PB2 (A), PB1 (B), PA (C) and NP (D) genes of the H9N2 influenza viruses analyzed.

Trees were generated on the basis of the following gene sequences: nucleotides 28–2307 (2279 bp, PB2), 40–1473 (1434 bp, PB1), 25–2175 (2151 bp, PA) and 46–1542 (1497 bp, NP). The PB2 and NP trees are rooted to A/Equine/Praque/1/56(H7N7), and the PB1 and PA trees to A/Equine/London/1416/73(H7N7). The other information was described in Fig. 1.

Figure 3. Replication of H9N2 viruses in tissues of the infected mice and chickens.

Mice and chickens were inoculated i.n. with Ck/HB/4/08 virus at a dose of 10⁶ EID₅₀ (mice), or 10⁷ EID₅₀ (chickens). Tissues were collected on 5 days p.i., and the virus was titrated in embryonated eggs from initial dilutions of 1∶10. Mean viral titers based on three mice or chickens per group are expressed as log₁₀ EID₅₀ per 10 milligrams wet tissues ± SD. Virus is below the detectable level at dilution of ≥10⁻¹. Open bars, chicken group; solid bars, mouse group.

Figure 4. Body weight and temperature of mice after H9N2 viral infection.

Mice were inoculated i.n. with 10⁶ EID₅₀ of Ck/HB/4/08 H9N2 viruses. Body weight (A) and temperature (B) were monitored daily for a 14-day observation period. Body weight was expressed as a percentage of the initial value. Data represent the mean of at least three mice of each group.

Figure 5. Survival percentage of mice after H9N2 viral infection.

Groups of mice (n = 14) were inoculated i.n. with each of six H9N2 viruses at a dose of 10⁷ EID₅₀ (A), 10⁶ EID₅₀ (B), 10⁵ EID₅₀ (C) or 10⁴ EID₅₀ (D), respectively. Percent survival was observed daily for 14 days.