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. 2021 Aug 25;95(18):e0044521.
doi: 10.1128/JVI.00445-21. Epub 2021 Aug 25.

Preferential Selection and Contribution of Non-Structural Protein 1 (NS1) to the Efficient Transmission of Panzootic Avian Influenza H5N8 Virus Clades 2.3.4.4A and B in Chickens and Ducks

Claudia Blaurock  1 , Angele Breithaupt  2 , David Scheibner  1 , Ola Bagato  1   3 , Axel Karger  1 , Thomas C Mettenleiter  4 , Elsayed M Abdelwhab  1
Affiliations

Preferential Selection and Contribution of Non-Structural Protein 1 (NS1) to the Efficient Transmission of Panzootic Avian Influenza H5N8 Virus Clades 2.3.4.4A and B in Chickens and Ducks

Claudia Blaurock et al. J Virol. .

Abstract

Highly pathogenic avian influenza virus H5N8 clade 2.3.4.4 caused outbreaks in poultry at an unprecedented global scale. The virus was spread by wild birds in Asia in two waves: clade 2.3.4.4A in 2014/2015 and clade 2.3.4.4B from 2016 up to today. Both clades were highly virulent in chickens, but only clade B viruses exhibited high virulence in ducks. Viral factors which contribute to virulence and transmission of these panzootic H5N8 2.3.4.4 viruses are largely unknown. The NS1 protein, typically composed of 230 amino acids (aa), is a multifunctional protein which is also a pathogenicity factor. Here, we studied the evolutionary trajectory of H5N8 NS1 proteins from 2013 to 2019 and their role in the fitness of H5N8 viruses in chickens and ducks. Sequence analysis and in vitro experiments indicated that clade 2.3.4.4A and clade 2.3.4.4B viruses have a preference for NS1 of 237 aa and 217 aa, respectively, over NS1 of 230 aa. NS217 was exclusively seen in domestic and wild birds in Europe. The extension of the NS1 C terminus (CTE) of clade B virus reduced virus transmission and replication in chickens and ducks and partially impaired the systemic tropism to the endothelium in ducks. Conversely, lower impact on fitness of clade A virus was observed. Remarkably, the NS1 of clade A and clade B, regardless of length, was efficient in blocking interferon (IFN) induction in infected chickens, and changes in the NS1 C terminus reduced the efficiency for interferon antagonism. Together, the NS1 C terminus contributes to the efficient transmission and high fitness of H5N8 viruses in chickens and ducks. IMPORTANCE The panzootic H5N8 highly pathogenic avian influenza viruses of clade 2.3.4.4A and 2.3.4.4B devastated the poultry industry globally. Clade 2.3.4.4A was predominant in 2014/2015 while clade 2.3.4.4B was widely spread in 2016/2017. The two clades exhibited different pathotypes in ducks. Virus factors contributing to virulence and transmission are largely unknown. The NS1 protein is typically composed of 230 amino acids (aa) and is an essential interferon (IFN) antagonist. Here, we found that the NS1 protein of clade 2.3.4.4A preferentially evolved toward long NS1 with 237 aa, while clade 2.3.4.4B evolved toward shorter NS1 with 217 aa (exclusively found in Europe) due to stop codons in the C terminus (CTE). We showed that the NS1 CTE of H5N8 is required for efficient virus replication, transmission, and endotheliotropism in ducks. In chickens, H5N8 NS1 evolved toward higher efficiency to block IFN response. These findings may explain the preferential pattern for short NS1 and high fitness of the panzootic H5N8 in birds.

Keywords: H5N8; NS1; clade 2.3.4.4; ducks; evolution; highly pathogenic avian influenza virus; interferon antagonism; transmission; virulence.

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Figures

FIG 1
FIG 1
Evolution of NS1 of clade 2.3.4.4A and 2.3.4.4B H5N8 viruses. NS gene sequences were retrieved from GISAID and were aligned using MAFFT. A midpoint-rooted phylogenetic tree was generated by MrBayes in Toplai v2. Two independent runs of 1,000,000 replicates and 10% burn-in were used. Shown is the phylogenetic relatedness of NS segment allele A of representative viruses. Clade A and clade B viruses used in this study are highlighted in gray. Viruses in black font have NS230, those in red have NS217, and those in blue have NS237. The putative ancestors are shown in boldface. Similar topology was obtained using neighbor-joining (NJ) and maximum-likelihood (ML) trees (data not shown) (A). R was used to determine the temporal distribution of NS1 length in sequences collected between 2013 and 2018 (B). Recombinant H5N8 clade A and clade B 2.3.4.4 viruses were generated carrying different NS1 CTEs. Clade A and B wild-type viruses have 237 and 217 aa, respectively. Dark red circles indicate point mutations in clade A compared to clade B. Red triangles indicate stop codons (C).
FIG 2
FIG 2
Preferential selection and impact of NS1 CTE on expression and replication of H5N8 viruses in cell culture. Preferential selection of NS1 was studied by cotransfection of cells for 2 days and propagation in ECE. Plaques were randomly selected, and NS1 was subjected to Sanger sequencing after RNA extraction and amplification of the NS1. The transfection was run in two separate rounds (A). Expression of NS1 4 h after infection of CEK cells with an MOI of 0.1. NS1 was detected by rabbit polyclonal NS1 antibodies, NP was detected by polyclonal NP rabbit antibody, and β-actin was detected with a commercial monoclonal antibody. Similar results were obtained at 8 and 24 hpi (data not shown). The signal above the NP band (∼70 kDa) is a nonspecific band (B). Numbers at left are molecular masses in kilodaltons (B). Replication of different viruses at an MOI of 0.001 at indicated time points in primary embryo kidney cells obtained from chickens (CEK) (C and D) or ducks (DEK) (E and F). Virus titers were determined by plaque assay in MDCKII cells. Statistical significance: *, P < 0.05.
FIG 3
FIG 3
The impact of NS1 on virus excretion and endotheliotropism in chickens. Virus excretion determined in oropharyngeal and cloacal swabs in inoculated chickens 2 dpi using quantitative RT-qPCR was expressed as log10 PFU/ml (eq.) (A and B). Endotheliotropism was determined by IHC. Scores are as follows: 0, negative; 1, focal to oligofocal; 2, multifocal; 3, coalescing foci or diffuse labeling. Dots represent individual animals. Bar shows median with interquartile range (C and D). Statistical significance: *, P < 0.05; **, P < 0.01.
FIG 4
FIG 4
The impact of NS1 on virulence, transmission, virus excretion, and endotheliotropism in ducks. Survival curves were generated using Kaplan-Meier analysis for clade A-inoculated (A) and contact (B) ducks and clade B-inoculated (C) and contact (D) ducks. Virus excretion determined in oropharyngeal and cloacal swabs in ducks inoculated with clade A (E) or clade B (F) viruses using quantitative RT-qPCR was expressed as log10 PFU/ml (eq.) in inoculated ducks. Viral tropism in ducks was determined by IHC at 2 days after inoculation of ducks with clade A (G) or clade B (H) viruses. Scores are as follows: 0, negative; 1, focal to oligofocal; 2, multifocal; 3, coalescing foci or diffuse labeling. GIT, gastrointestinal tract. Dots represent individual animals; bar shows median with interquartile range. Endothelium scores indicate the maximum score found in all tissues affected. Statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
FIG 5
FIG 5
Immunohistopathological detection of Matrix antigen in selected organs in ducks. Organs were obtained from n = 3 inoculated ducks per group, euthanized 4 dpi. Scores indicate the semiquantitative amount of viral antigen found within the group; the number of animals tested positive per group is in parentheses. Immunohistochemistry, ABC method using anti-Matrix protein antibody, AEC chromogen (red-brown), hematoxylin (blue) counterstain. Bar = 50 μm (liver and spleen) or 100 μm (brain and lung). Note inset of panel N representatively showing endotheliotropism.
FIG 6
FIG 6
Interferon induction in lungs of chickens and ducks inoculated with clade A and B H5N8 viruses. Shown are the fold changes in IFN levels in the lungs of chickens and ducks inoculated with clade A and B H5N8 viruses 2 dpi. Tissues were collected, weighed (wt/vol), and homogenized. The mRNAs of IFN-α, IFN-β, or IFN-γ were measured by generic RT-qPCR from 3 birds in each group. Normalization was done using 28S rRNA transcripts. Results were calculated using the 2^−(ΔΔCT) method and expressed as fold change of normalized samples compared to samples obtained from noninfected birds (n = 3). Statistical significance: *, P < 0.05; **, P < 0.01.
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