Genetics, receptor binding property, and transmissibility in mammals of naturally isolated H9N2 Avian Influenza viruses

Abstract

H9N2 subtype influenza viruses have been detected in different species of wild birds and domestic poultry in many countries for several decades. Because these viruses are of low pathogenicity in poultry, their eradication is not a priority for animal disease control in many countries, which has allowed them to continue to evolve and spread. Here, we characterized the genetic variation, receptor-binding specificity, replication capability, and transmission in mammals of a series of H9N2 influenza viruses that were detected in live poultry markets in southern China between 2009 and 2013. Thirty-five viruses represented 17 genotypes on the basis of genomic diversity, and one specific "internal-gene-combination" predominated among the H9N2 viruses. This gene combination was also present in the H7N9 and H10N8 viruses that have infected humans in China. All of the 35 viruses preferentially bound to the human-like receptor, although two also retained the ability to bind to the avian-like receptor. Six of nine viruses tested were transmissible in ferrets by respiratory droplet; two were highly transmissible. Some H9N2 viruses readily acquired the 627K or 701N mutation in their PB2 gene upon infection of ferrets, further enhancing their virulence and transmission in mammals. Our study indicates that the widespread dissemination of H9N2 viruses poses a threat to human health not only because of the potential of these viruses to cause an influenza pandemic, but also because they can function as "vehicles" to deliver different subtypes of influenza viruses from avian species to humans.

Figure 1. Genetic relationships among the HA genes and genotype evolution of H9N2 viruses.

(A) Phylogenetic tree of HA. The tree was based on nucleotides (nt) 128 to 1540 and rooted to A/Duck/Alberta/60/76(H12N5). Sequences of viruses with names in black were downloaded from available databases; viruses with names in other colors were sequenced in this study. Abbreviations are as follows: Br, Brambling; CAn, Canine; CK, Chicken; DK, Duck; Env, Environment; GS, Goose; Pg, Pigeon; QA, Quail; SW, Swine; Ty, Turkey; AH, Anhui; BJ, Beijing; CA, California; CQ, Chongqing; DH, Donghu; FJ, Fujian; GD, Guangdong; GX, Guangxi; GZ, Guangzhou; HZ, Hangzhou; HN, Henan; HK, Hong Kong; HuB, Hubei; HuN, Hunan; JS, Jiangsu; SD, Shandong; SH, Shanghai; TZ, Taizhou; WI, Wisconsin; XZ, Xuzhou; YZ, Yangzhou; YN, Yunnan; ZJ, Zhejiang. (B) Genotypes of the H9N2 viruses. The eight gene segments are indicated at the top of each bar.

Figure 2. Characterization of the receptor-binding properties of H9N2 viruses.

The binding of the viruses to two different biotinylated glycans (α-2, 3 glycan, blue; α-2, 6 glycan, pink) was tested. The data shown are the means of three repeats; the error bars indicate the standard deviations. (A) CK/GX/C1435/12. (B) CK/SH/SC197/13. (C) DK/ZJ/C2046/12. (D) CK/ZJ/SC324/13. (E) CK/ZJ/C1219/10. (F) CK/JS/C4258/12. (G) CK/CQ/C1258/11. (H) CK/HuB/C4196/09. (I) CK/HuN/C4136/10. (J) rCK/GX/9/99. (K) rCK/GX/9/99-HAT155I. (L) rCK/GX/9/99-HAN183H.

Figure 3. Replication and virulence of H9N2 viruses in mice.

Virus titers in organs of mice on day 3 p.i. with 10⁶ EID₅₀ of test virus. Data shown are the mean titers from three mice; the error bars indicate the standard deviations. *, mice inoculated with CK/ZJ/C1219/10-PB2/627K virus died before day 8 p.i. The dashed line indicates the lower limit of detection.

Figure 4. Replication of H9N2 viruses in ferrets.

(A) CK/GX/C1435/12. (B) CK/SH/SC197/13. (C) DK/ZJ/C2046/12. (D) CK/ZJ/SC324/13. (E) CK/ZJ/C1219/10. (F) CK/JS/C4258/12. (G) CK/CQ/C1258/11. (H) CK/HuB/C4196/09. (I) CK/HuN/C4136/10. (J) CK/ZJ/C1219/10-PB2/627K. (K) CK/ZJ/C1219/10-PB2/701N. Each color bar represents the virus titer from an individual animal. The dashed black lines indicate the lower limit of detection.

Figure 5. Histological lesions caused by H9N2 viruses in the lungs of ferrets.

Ferrets were euthanized on day 4 p.i. with 10⁶EID₅₀ of test virus, and the lungs were collected for pathological study. The lungs of CK/ZJ/C1219/10 virus-inoculated animal showed only mild histopathological changes (H&E staining,) (A), whereas the lungs of CK/GX/C1435/12 (B), CK/JS/C4258/12 (C), CK/ZJ/C1219/10-PB2/627K (D), and CK/ZJ/C1219/10-PB2/701N (E) virus-inoculated ferrets showed severe pathological lesions (H&E staining) Viral antigen was detected in the epithelial cells of bronchus and alveoli by means of immunohistochemical (IHC) staining (F, from the lung samples of a ferret inoculated with CK/JS/C4258/12 virus). Images A–E were taken at ×100 magnification; Image F was taken at ×200 magnification.

Figure 6. Respiratory droplet transmission of H9N2 viruses in ferrets.

(A) CK/GX/C1435/12. (B) CK/SH/SC197/13. (C) DK/ZJ/C2046/12. (D) CK/ZJ/SC324/13. (E) CK/ZJ/C1219/10. (F) CK/JS/C4258/12. (G) CK/CQ/C1258/11. (H) CK/HuB/C4196/09. (I) CK/HuN/C4136/10. (J) CK/JS/C4258/12 (replicate). (K) CK/ZJ/C1219/10-PB2/627K. (L) CK/ZJ/C1219/10-PB2/701N. Each color bar represents the virus titer from an individual animal. The dashed black lines indicate the lower limit of detection.