Assessing compatibility and viral fitness between poultry-adapted H9N2 and wild bird-derived neuraminidases

Abstract

Exchange of viral segments between one or more influenza virus subtypes can contribute to a shift in virulence and adaptation to new hosts. Among several influenza subtypes, H9N2 is widely circulating in poultry populations worldwide and has the ability to infect humans. Here, we studied the reassortant compatibility between chicken H9N2 with N1-N9 gene segments of wild bird origin, either with an intact or truncated stalk. Naturally occurring amino acid deletions in the NA stalk of the influenza virus can lead to increased virulence in both mallard ducks and chickens. Our findings show extended genetic compatibility between chicken H9Nx gene segments and the wild-bird NA with and without 20 amino acid stalk deletion. Replication kinetics in avian, mammalian and human cell lines revealed that parental chH9N2 and rH9N6 viruses with intact NA-stalk replicated significantly better in avian DF1 cells compared to human A549 cells. After introducing a stalk deletion, an enhanced preference for replication in mammalian and human cell lines could be observed for rH9N2_Δ(H6), rH9N6_Δ and rH9N9_Δ compared to the parental chH9N2 virus. This highlights the potential emergence of novel viruses with variable phenotypic traits, warranting the continuous monitoring of H9N2 and co-circulating subtypes in avian hosts.

Figure 1

HYPERLINK "sps:id::fig1||locator::gr1||MediaObject::0" RAxML midpoint tree based on wild bird and poultry-adapted Neuraminidase sequences (N1–N9). The scale bar represents nucleotide substitutions per site, and branch labels show the bootstrap value in percentage. Virus isolates used in this study are highlighted in bold.

Figure 2

Replication efficiency of the rH9Nx reassortants with intact neuraminidase stalks in DF-1 (orange) and A549 (blue). The log₁₀(TCID₅₀) value at time point 0 h is based on the starting TCID₅₀ value. Error bars represent the standard error of the mean.

Figure 3

Replication efficiency of the rH9Nx reassortants with a 20 aa neuraminidase stalk deletion in DF-1 (orange) and A549 (blue). The log₁₀(TCID₅₀) value at time point 0 h is based on the starting TCID₅₀ value. Error bars represent the standard error of the mean (SEM).

Figure 4

NA activity of the rH9Nx reassortants with and without a 20 aminoacid stalk deletion. Graph demonstrates the Relative Luminescence Unit at given HAU titers. Errorbars represent the standard error of the mean (SEM).