Evidence of expanded host range and mammalian-associated genetic changes in a duck H9N2 influenza virus following adaptation in quail and chickens

Abstract

H9N2 avian influenza viruses continue to circulate worldwide; in Asia, H9N2 viruses have caused disease outbreaks and established lineages in land-based poultry. Some H9N2 strains are considered potentially pandemic because they have infected humans causing mild respiratory disease. In addition, some of these H9N2 strains replicate efficiently in mice without prior adaptation suggesting that H9N2 strains are expanding their host range. In order to understand the molecular basis of the interspecies transmission of H9N2 viruses, we adapted in the laboratory a wildtype duck H9N2 virus, influenza A/duck/Hong Kong/702/79 (WT702) virus, in quail and chickens through serial lung passages. We carried out comparative analysis of the replication and transmission in quail and chickens of WT702 and the viruses obtained after 23 serial passages in quail (QA23) followed by 10 serial passages in chickens (QA23CkA10). Although the WT702 virus can replicate and transmit in quail, it replicates poorly and does not transmit in chickens. In contrast, the QA23CkA10 virus was very efficient at replicating and transmitting in quail and chickens. Nucleotide sequence analysis of the QA23 and QA23CkA10 viruses compared to the WT702 virus indicated several nucleotide substitutions resulting in amino acid changes within the surface and internal proteins. In addition, a 21-amino acid deletion was found in the stalk of the NA protein of the QA23 virus and was maintained without further modification in the QA23CkA10 adapted virus. More importantly, both the QA23 and the QA23CkA10 viruses, unlike the WT702 virus, were able to readily infect mice, produce a large-plaque phenotype, showed faster replication kinetics in tissue culture, and resulted in the quick selection of the K627 amino acid mammalian-associated signature in PB2. These results are in agreement with the notion that adaptation of H9 viruses to land-based birds can lead to strains with expanded host range.

Figure 1. Replication of H9N2 duck, quail- or chicken-adapted viruses.

A) Quail and B) chickens were infected with the indicated doses (in EID₅₀) of WT702, QA23, or QA23CkA10 viruses (set of birds infected with given virus are marked as +++). Each bar corresponds to virus titers in lung homogenates of individual birds collected at 3 dpi. C) Chickens infected with the indicated dose of WT702, QA23, or QA23CkA10 viruses (set of birds infected with given virus are marked as +++). At 3 dpi, chickens were sacrificed and intestine collected to determine virus titers. * indicates no virus detected in the undiluted samples. Virus titers presented as log₁₀EID₅₀/mL.

Figure 2. Plaque morphology and growth kinetics of H9N2 viruses in MDCK and CEK cells.

A) Confluent monolayers of MDCK or CEK cells were infected with the corresponding viruses and maintained at 37°C in agar-maintenance medium supplemented with TPCK-trypsin. Cells were stained with crystal violet at 4 (MDCK) and 2 (CEK) dpi. B) Growth kinetics in confluent monolayer of MDCK (top panels) or CEK (bottom panels) infected with WT702 (open bar), QA23 (shaded bar) or QA23CkA10 (black bar) viruses at an input m.o.i of 0.001. Infected cells were maintained at 37°C in maintenance medium supplemented with TPCK-trypsin. Culture supernatants were collected at different hpi and virus titers measured by HA assay (left panels) or TCID₅₀ in CEK cells (right panels).

Figure 3. Comparison of nucleotide sequences of the PB2 gene near positions encoding for amino acid 627 between duck, quail- and chicken-adapted viruses before and after a single round of infection in mice.

Alignment of electropherograms from PB2 sequences obtained from the WT702, QA23, and QA23CkA10 viruses before and after viruses were isolated from the lungs of mice. The arrowhead indicates the position of nucleotide change from G to A that generates the E627K mutation in PB2. E, K, or K/E, on the right of each electropherogram correspond to the predicted encoded amino acid for position 627 in the PB2 open reading frame. PP, sequence derived from plaque purified virus; ML, sequence derived from virus in mouse lung; ML-Egg, sequence derived from virus isolated from mouse lungs and amplified once in embryonated eggs.

Figure 4. Body weight changes and lung virus titers in mice infected with H9N2 viruses.

A) Groups of 4 mice were inoculated intranasally with 10⁷ EID₅₀ of QA23ML-3 Egg (black squares) or QA23 PP (open squares) viruses. Body weight and clinical signs of disease were monitored for 14 dpi. Data presented correspond to average body weight changes in each group with corresponding standard deviations. †, one mouse infected with the QA23ML-3 Egg was humanely sacrificed due to severe disease signs. B) Virus titers in lung homogenates of mice (4/group) infected with 10⁷ EID₅₀ of the WT702, QA23CkA10 PP, QA23 PP and QA23ML-3 Egg viruses as indicated. E627 and K627 correspond to the encoded amino acid position 627 in PB2.