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. 2022 Jul 13;96(13):e0014922.
doi: 10.1128/jvi.00149-22. Epub 2022 Jun 7.

Genetic Determinants for Virulence and Transmission of the Panzootic Avian Influenza Virus H5N8 Clade 2.3.4.4 in Pekin Ducks

David Scheibner  1 , Angele Breithaupt  2 , Christine Luttermann  3 , Claudia Blaurock  1 , Thomas C Mettenleiter  4 , Elsayed M Abdelwhab  1
Affiliations

Genetic Determinants for Virulence and Transmission of the Panzootic Avian Influenza Virus H5N8 Clade 2.3.4.4 in Pekin Ducks

David Scheibner et al. J Virol. .

Abstract

Waterfowl is the natural reservoir for avian influenza viruses (AIV), where the infection is mostly asymptomatic. In 2016, the panzootic high pathogenicity (HP) AIV H5N8 of clade 2.3.4.4B (designated H5N8-B) caused significant mortality in wild and domestic ducks, in stark contrast to the predecessor 2.3.4.4A virus from 2014 (designated H5N8-A). Here, we studied the genetic determinants for virulence and transmission of H5N8 clade 2.3.4.4 in Pekin ducks. While ducks inoculated with recombinant H5N8-A did not develop any clinical signs, H5N8-B-inoculated and cohoused ducks died after showing neurological signs. Swapping of the HA gene segments did not increase virulence of H5N8-A but abolished virulence and reduced systemic replication of H5N8-B. Only H5N8-A carrying H5N8-B HA, NP, and NS with or without NA exhibited high virulence in inoculated and contact ducks, similar to H5N8-B. Compared to H5N8-A, HA, NA, NS, and NP proteins of H5N8-B possess peculiar differences, which conferred increased receptor binding affinity, neuraminidase activity, efficiency to inhibit interferon-alpha induction, and replication in vitro, respectively. Taken together, this comprehensive study showed that HA is not the only virulence determinant of the panzootic H5N8-B in Pekin ducks, but NP, NS, and to a lesser extent NA were also necessary for the exhibition of high virulence in vivo. These proteins acted synergistically to increase receptor binding affinity, sialidase activity, interferon antagonism, and replication. This is the first ad-hoc study to investigate the mechanism underlying the high virulence of HPAIV in Pekin ducks. IMPORTANCE Since 2014, several waves of avian influenza virus (AIV) H5N8 of clade 2.3.4.4 occurred globally on unprecedented levels. Unlike viruses in the first wave in 2014-2015 (H5N8-A), viruses in 2015-2016 (H5N8-B) exhibited unusually high pathogenicity (HP) in wild and domestic ducks. Here, we found that the high virulence of H5N8-B in Pekin ducks could be attributed to multiple factors in combination, namely, hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), and nonstructural protein 1 (NS1). Compared to H5N8-A, H5N8-B possesses distinct genetic and biological properties including increased HA receptor-binding affinity and neuraminidase activity. Likewise, H5N8-B NS1 and NP were more efficient to inhibit interferon induction and enhance replication in primary duck cells, respectively. These results indicate the polygenic trait of virulence of HPAIV in domestic ducks and the altered biological properties of the HPAIV H5N8 clade 2.3.4.4B. These findings may explain the unusual high mortality in Pekin ducks during the panzootic H5N8 outbreaks.

Keywords: H5N8; HPAI; ducks; hemagglutinin; highly pathogenic avian influenza virus; neuramindase; non-structural protein NS1; nucleoprotein; virulence determinants.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Recombinant H5N8 clade 2.3.4.4 viruses generated in this study. Blue and red colors refer to the gene segments of clade 2.3.4.4 avian influenza viruses of clade A and B, respectively. Recombinant H5N8-A viruses carrying different gene segments (subscript letters) of H5N8-B virus were generated by transfecting cell culture and propagation in embryonated chicken eggs. Titers were determined in plaque assay and expressed as PFU per 1 mL (PFU/mL).
FIG 2
FIG 2
Detection of viral RNA in swab samples collected from inoculated and cohoused ducks. Viral RNA was detected using RT-qPCR targeting the NP gene. Oropharyngeal and cloacal swabs from inoculated (A, B) and cohoused ducks (C, D) were collected at 4 dpi and were individually tested. Standard curves using serial dilutions of H5N8-A were generated in each 96-well plate. Samples were quantified by plotting the Ct-value to the dilution of H5N8-A and expressed as the equivalent logarithm of virus PFU per 1 mL (PFU/mL eq). Shown are the individual titers and bars indicating the mean and standard deviations of positive samples. Dashed line indicates the predictive detection limit. Asterisks indicate significance difference at P value < 0.05.
FIG 3
FIG 3
Tropism and histopathologic changes in Pekin ducks infected with recombinant H5N8 clade 2.3.4.4 viruses. Samples were collected from ducks at 4 dpi, and distribution of matrix-1 antigen using immunohistochemistry (IHC) (A) and histopathologic changes (B) were determined for each group. The heatmap color corresponds to the maximum score specified per group and indicates the number of positive/test animals for each organ. For the IHC, the viral antigen was semiquantitatively scored: 0 = negative; 1 = focal or oligofocal, 2 = multifocal, 3 = coalescing, 4 = diffuse immunoreactive cells (A). For histopathologic changes, necrotizing inflammation in the brain, heart, liver, and immune organs were semiquantitatively scored: 0 = no changes, 1 = focal or oligofocal, 2 = multifocal, 3 = coalescing, 4 = diffuse. The cellularity in immune organs was scored with 0 = no changes, 1 = minimal decrease, 2 = mild decrease, 3 = moderate decrease, 4 = severe decrease. The heatmap color corresponds to the maximum score specified per group and indicates the number of positive/test animals for each organ.
FIG 4
FIG 4
Detection of H5N8 matrix-1 protein in different tissues of inoculated ducks. Shown is the influenza A matrix-1 protein (bright red staining) in the brain, heart, and liver of inoculated ducks using immunohistochemistry, 4 dpi. Scale bars indicate 50 μm. Note the endotheliotropism for H5N8-B exemplarily shown in the brain and heart (inlay). Antigen labeling for H5N8-A_BHA-NS corresponds to H5N8-A_BHA-NA (the latter not shown).
FIG 5
FIG 5
Phylogenetic relatedness of different genes of H5N8 2.3.4.4 clades A and B from Eurasia and North America in 2014–2017. Shown are the cladograms of different genes of clade A and B H5N8 viruses from Eurasia and North America from 2014 to 2017. The sequences of clade A viruses from North America (light green) and Eurasia (yellow) in 2014/2015 and clade B viruses from Eurasia in 2016/2017 (gray) are shown (Table S1). Recombinant viruses used in this study are depicted in blue (clade A) and red (clade B). Sequences of the recombinant H5N8-A and H5N8-B viruses as well as a representative strain (A/gyrfalcon/Washington/41088-6/2014) from North America were selected by nucleotide blasting in GISAID database. Primary phylogenetic trees of the first 200 closely related sequences were generated by IQTree. Representative sequences were further selected to construct midpoint rooted trees by IQTree and MrBayes provided by Topali and further edited for publishing using FigTree and Inkscape free software. Bootstrap values are indicated in the main branches, and bars indicate genetic distances.
FIG 6
FIG 6
Genetic differences of the HA and NA of clades A and B H5N8 viruses in this study and the biological impact on virus fitness in vitro. Shown is a graph for different domains of the HA protein (A) showing the signal peptide and HA1 (orange) and HA2 (cyan) subunits connected by the proteolytic polybasic cleavage site (pCS) (blue). Amino acid differences between H5N8-A and H5N8-B viruses are indicated in red lines. Ag-epitope, antigenic epitopes; TMD, transmembrane domain (A). Predicted tertiary structure of the HA depicting HA1 (orange) and HA2 (cyan) subunits and the pCS (blue ribbons) is shown. Amino acid differences between H5N8-A and H5N8-B viruses are shown in red spheres (B). Cleavability of the HA0 of H5N8-A and H5N8-B into HA1 and HA2 polypeptides in duck cells at MOI of 1 was detected using anti-H5 antibodies in standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot procedures. “NC” refers to the negative control noninfected cells (C). Receptor binding affinity of indicated viruses (adjusted to 32 HA units) against different dilutions of α-2,3 sialic acid labeled HRP-fetuin (x axis) was assessed using solid-phase assay. The absorbance or optical density (OD) indicate binding affinity at 450 nm wavelength was measured using Tecan ELISA Reader (y axis). Data presented are mean ± standard deviation of two quadruplicate independent experiments (D). A graph for the NA protein containing the N-terminal (N), transmembrane (TMD), and stalk and head domains is shown (E). Amino acid differences (red spheres) are imposed on the predicted tertiary structure of the NA head domain tetramers. The sialidase functional and frame sites are shown in blue sticks (F). NA activity (y axis) against MUNANA substrate is shown as relative fluorescence units (RFU) (G), or against fetuin are shown as absorbance values at 490 nm wavelength (H). For biosafety reasons, the NA activity assays were done using different dilutions of recombinant PR8 H1N1 carrying NA from H5N8-A or H5N8-B (x axis). Results are expressed as average and standard deviation of two duplicate independent experiments.
FIG 7
FIG 7
Genetic differences of the NP and NS1 of clades A and B H5N8 viruses in this study and the biological impact on virus fitness in vitro. Shown is a graph for NP protein (A) illustrating the two PB2-binding domains (orange), RNA-binding domain (cyan), and NP-NP interaction domain (magenta). The 5 amino acid differences between H5N8-A and H5N8-B viruses are indicated in red lines in the graph (A) or red spheres in the predicted tertiary structure of the NP (B). Replication kinetics of indicated viruses were done in primary duck kidney cells infected at MOI of 0.001 for 8 and 24 h. Virus titration was done by plaque assay in MDCKII cells, and the dashed line is the assumed detection limit of the plaque assay. The results are expressed as mean and standard deviation of PFU/mL of three independent experiments (C). A graph for the NS1 protein showing the RNA binding (cyan) and effector domains (yellow) connected by a linker (gray). Variations in the stop codons in the C-terminus resulted in NS1 of 237 or 217 aa length in H5N8-A and H5N8-B, respectively (D). Amino acid differences between H5N8-A and H5N8-B NS1 are indicated in red lines in the graph (D) or red spheres on the predicted tertiary structure of NS1 dimers (ribbon and sticks) (E). The efficiency of different NS1 to block IFN-α and IFN-β induction was studied in DF1 (F) and HEK293T (G) cells using luciferase assay. ch, chicken; hu, human. The results are expressed as a fold change of IFN induction relative to the signal induced by the given trigger for the empty vector control (y axis). Asterisks indicate significant differences at P value less than 0.05. Ctrl, empty vector with trigger; -, empty vector without trigger.
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