Replication and transmission of H9N2 influenza viruses in ferrets: evaluation of pandemic potential

Abstract

H9N2 avian influenza A viruses are endemic in poultry of many Eurasian countries and have caused repeated human infections in Asia since 1998. To evaluate the potential threat of H9N2 viruses to humans, we investigated the replication and transmission efficiency of H9N2 viruses in the ferret model. Five wild-type (WT) H9N2 viruses, isolated from different avian species from 1988 through 2003, were tested in vivo and found to replicate in ferrets. However these viruses achieved mild peak viral titers in nasal washes when compared to those observed with a human H3N2 virus. Two of these H9N2 viruses transmitted to direct contact ferrets, however no aerosol transmission was detected in the virus displaying the most efficient direct contact transmission. A leucine (Leu) residue at amino acid position 226 in the hemagglutinin (HA) receptor-binding site (RBS), responsible for human virus-like receptor specificity, was found to be important for the transmission of the H9N2 viruses in ferrets. In addition, an H9N2 avian-human reassortant virus, which contains the surface glycoprotein genes from an H9N2 virus and the six internal genes of a human H3N2 virus, showed enhanced replication and efficient transmission to direct contacts. Although no aerosol transmission was observed, the virus replicated in multiple respiratory tissues and induced clinical signs similar to those observed with the parental human H3N2 virus. Our results suggest that the establishment and prevalence of H9N2 viruses in poultry pose a significant threat for humans.

Figure 1. Replication and direct contact transmission of H9N2 viruses.

Ferrets were inoculated intranasally (i.n.) with 10⁶ TCID₅₀ of H9N2 viruses RGWF10 (A), Dk/HK/Y280/97 (B), RGQa88 (C), Ck/HK/SF3/99 (D), and Ck/Jordan/554/03 (E). Twenty-four hours later, one naïve ferret (direct contact) was added to the same cage as each of the infected ferrets. Nasal washes were collected daily and were titrated in MDCK cells. Black, white and gray bars represent individual ferrets sampled and the amount of viral shedding at different days pi. The titers are expressed as log₁₀ values of TCID₅₀/ml with the limit of detection at 0.699 log₁₀TCID₅₀/ml. The dotted line was arbitrarily set at <0.3 log₁₀TCID₅₀/ml in order to represent samples below the detection limit. L and Q correspond to Leu226 and Gln226, respectively in the HA RBS.

Figure 2. Aerosol transmission of H9N2 and H3N2 viruses.

Ferrets were inoculated i.n. with 10⁶ TCID₅₀ of RGWF10 or RGMemphis98 virus. Twenty-four hours later, one naïve ferret was added to each infected ferret to serve as direct contact, and another ferret was placed into an adjacent cage separated by a wire mesh to serve as aerosol contact. Nasal washes were collected daily and were titrated in MDCK cells. (A) RGWF10 infected and direct contacts. (B) RGWF10 aerosol contacts. (C) RGMemphis98 inoculated and direct contacts. (D) RGMemphis98 aerosol contacts.

Figure 3. Replication and transmission of mutant H9N2 viruses.

Ferrets were inoculated i.n. with 10⁶ TCID₅₀ of mWF10 (Gln226) or mQa88 (Leu226) virus. Twenty-four hours later, contact ferrets were introduced as described above. Nasal washes were collected daily and were titrated by TCID₅₀. (A) mWF10 infected and direct contacts. (B) mWF10 aerosol contacts. (C) mQa88 infected and direct contacts. L and Q correspond to Leu226 and Gln226, respectively in the HA RBS.

Figure 4. Effect of Leu226 or Gln226 mutations on receptor specificity of H9N2 viruses.

The importance of amino acid 226 of HA in receptor specificity was confirmed by site-directed mutagenesis followed by glycan microarray analysis. Viruses with a natural Leu226 (A: RGWF10 L) or Gln226Leu mutation (D: mQa88 L ) bind to human-type α2-6 sialosides (glycans 33 to 46), whereas viruses with Leu226Gln mutation (B: mWF10 Q) or natural Gln226 (C: RGQa88 Q) bind to avian-type α2-3 sialosides (glycans 1 to 32). Viruses were analyzed at hemagglutination titers of 128 per 50 µl. Allantoic fluid was used as negative control (E). Glycans 1–32 are avian-type α2-3 sialosides (light gray) and 33–46 are human-type α2-6 sialosides (dark gray). L and Q correspond to Leu226 and Gln226, respectively in the HA RBS. The complete structures of each sialoside are available upon request.

Figure 5. Receptor specificity of other wild type H9N2 isolates.

Other selected H9N2 isolates from 1977 to 1999 were assessed on the glycan microarray as previously described . Shown are results for Dk/HK/149/77, an isolate with Gln226 (Q), and three later isolates with Leu226 (L) Dk/Y280/97, Hu/HK/1073/99, and Qa/HK/NT16/99. Viruses were analyzed at a hemagglutination titer of 256 or 128 (Hu/HK/1073/99). L and Q correspond to Leu226 and Gln226, respectively in the HA RBS.

Figure 6. Recovery and plaque assay of an H9N2 avian-human reassortant virus.

(A) Diagram outlining gene segment exchange to create the reassortant virus. (B) Plaque morphology of the parental H3N2 virus RGMemphis98 (left), the parental H9N2 virus RGWF10 (center) and the 2WF10:6M98 reassortant virus (right).

Figure 7. Replication and transmission of the H9N2 avian-human reassortant virus.

Ferrets were inoculated i.n. with 10⁶ TCID₅₀ of 2WF10:6M98 virus. Twenty-four hours later, the direct and aerosol contacts were placed in the cages as described above. Nasal washes were collected daily and were titrated by TCID₅₀. (A) 2WF10:6M98 virus inoculated and direct contacts. (B) 2WF10:6M98 virus aerosol contacts.

Figure 8. Histopathology and virus distribution of H9N2 viruses in ferrets.

Two ferrets were inoculated i.n. with 10⁶ TCID₅₀ for each virus: mWF10, RGWF10 or 2WF10:6M98. At day 4 p.i, ferrets were euthanized and the tracheas and lungs were harvested for histological analysis. (A) Histopathological findings in the respiratory tract. Upper panel, tracheas: note the margination of neutrophils (▾) and mononuclear cells (↑) in a small vein in the 2WF10:6M98-infected trachea. Lower panel, lungs: note the severe inflammatory infiltration in the 2WF10:6M98-infected lung. (B) Tissue tropism in organs collected from ferrets inoculated with mWF10, RGWF10, or 2WF10:6M98 virus. OB, olfactory bulb. NT, nasal turbinate.